WO2023287269A1 - Bio-marker composition for diagnosing brain disease due to brain microvascular damage - Google Patents
Bio-marker composition for diagnosing brain disease due to brain microvascular damage Download PDFInfo
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- WO2023287269A1 WO2023287269A1 PCT/KR2022/095112 KR2022095112W WO2023287269A1 WO 2023287269 A1 WO2023287269 A1 WO 2023287269A1 KR 2022095112 W KR2022095112 W KR 2022095112W WO 2023287269 A1 WO2023287269 A1 WO 2023287269A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Definitions
- the present invention is a biomarker composition for diagnosing brain diseases caused by cerebral microvascular damage, and more specifically, brain diseases caused by cerebral microvascular damage by precisely classifying local areas of the brain based on MRI images and inducing degeneration of the blood-brain barrier only in those local areas.
- the blood-brain barrier is a barrier that exists between the brain and blood vessels, preventing foreign substances from entering the brain.
- the blood-brain barrier prevents foreign substances from entering the brain and protects the brain by accepting substances necessary for metabolism. For example, it prevents the invasion of bacteria or viruses in the brain, and facilitates glucose supply and oxygen-carbon dioxide exchange. Blood is filtered through the blood-brain barrier, and as a result, very few substances reach the brain, such as water or gas molecules, glucose, and certain fat-soluble substances.
- the blood-brain barrier has a shape that surrounds blood vessels with brain endothelial cells, and it is impossible for substances to pass between cells because the endothelial cells are in tight junctions with each other.
- the blood-brain barrier prevents the passage of not only various imaging agents for diagnosis, but also drugs when diseases occur in the brain, such as tumors, Alzheimer's disease, and Parkinson's disease, thereby hindering the development of brain-related diagnosis and treatment technologies.
- the inventors of the present invention irradiated ultrasonic energy to mice to induce degeneration of the blood-brain barrier to produce a mouse model of brain disease caused by brain microvascular damage, and the mouse Proteins isolated from the brain tissue of the model were analyzed to screen for biomarkers related to brain microvascular damage and encephalopathy due to blood-brain barrier degeneration. By confirming that it is a biomarker, the present invention was completed.
- An object of the present invention is to provide a biomarker composition for diagnosing brain diseases caused by brain microvascular damage, including any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn.
- Another object of the present invention is to provide a composition for diagnosing brain diseases caused by cerebral microvascular damage, comprising an agent for measuring the mRNA expression or protein activity level of any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn. is to do
- Another object of the present invention is to provide a kit for diagnosing brain diseases caused by brain microvascular damage comprising the above-described composition.
- Another object of the present invention is to provide a method for screening a pharmaceutical composition for preventing or treating brain diseases caused by brain microvascular damage, comprising the following steps. (a) treating the biological sample with any compound; (b) confirming the expression level or activity level of any one or more genes or proteins thereof selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn from the biological sample; and (c) comparing the expression level with the level of the gene or its expressed protein or activity level in a normal control group not treated with any compound.
- Another object of the present invention is to provide a method for producing an optimal BBB degenerated mouse model comprising the step of (a) treating the mouse with focused ultrasound at 0.25 to 0.27 MPa for 10 to 230 seconds.
- Another object of the present invention is to provide a method for producing a mildly damaged BBB degenerated mouse model comprising the step of (a) treating the mouse with focused ultrasound at 0.42 to 0.44 MPa for 10 to 230 seconds.
- the present invention provides a biomarker composition for diagnosing brain diseases caused by cerebral microvascular damage, including any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn. can do.
- the present invention can also provide a composition for diagnosing brain diseases caused by cerebral microvascular damage comprising an agent for measuring the mRNA expression or protein activity level of any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn. there is.
- the agent may be a primer or probe that specifically binds to any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn.
- the agent may be an antibody that specifically binds to a protein of one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn.
- the present invention can also provide a kit for diagnosing brain diseases caused by brain microvascular damage comprising the above-described composition.
- the present invention also relates to the use of a biomarker composition comprising at least one gene selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn for diagnosing brain diseases caused by brain microvascular damage and Tgfbi, Cst7 from biological samples. It is possible to provide a method for diagnosing brain diseases caused by damage to brain microvessels including; measuring the mRNA expression level or the protein activity level of the Angptl4 or Srgn gene.
- the present invention is also directed to a method for diagnosing brain diseases caused by brain microvascular damage comprising measuring the mRNA expression or protein activity level of any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn from a biological sample. How to provide information can be provided.
- the present invention may provide a method for screening a pharmaceutical composition for preventing or treating brain diseases caused by brain microvascular damage, comprising the following steps: (a) treating a biological sample with an arbitrary compound; (b) confirming the expression level or activity level of any one or more genes or proteins thereof selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn from the biological sample; and (c) comparing the expression level with the level of the gene or its expressed protein or activity level in a normal control group not treated with any compound.
- the present invention may provide a method for producing an optimal BBB degenerated mouse model comprising the step of (a) treating the mouse with focused ultrasound at 0.25 to 0.27 MPa for 10 to 230 seconds.
- the present invention may provide a method for producing a mildly damaged BBB degenerated mouse model comprising the step of (a) treating the mouse with focused ultrasound at 0.42 to 0.44 MPa for 10 seconds to 230 seconds.
- a mouse model of brain microvascular damage and brain disease caused by opening of the blood-brain barrier was prepared by irradiating a mouse with focused ultrasound.
- the degree of patency of the blood-brain barrier was controlled by adjusting the intensity of the focused ultrasound, and the control caused optimal and moderate damage, thereby producing an optimal BBB degenerated mouse model and a mild BBB degenerated mouse model did
- transcriptome sequencing analysis a biological analysis method, was performed to discover diagnostic markers for brain diseases caused by BBB degeneration. was confirmed to be diagnosable.
- Tgfbi, Cst7, Angptl4 and Srgn were increased in the blood of optimal BBB degeneration animal models and mild BBB degeneration animal models. Based on the above results, there is an effect of diagnosing brain diseases such as dementia, brain cancer, Parkinson's, concussion (TBI) or stroke caused by brain microvascular damage using Tgfbi, Cst7, Angptl4 or Srgn.
- brain diseases such as dementia, brain cancer, Parkinson's, concussion (TBI) or stroke caused by brain microvascular damage using Tgfbi, Cst7, Angptl4 or Srgn.
- FIG. 1 is a schematic diagram of a method for manufacturing an animal model of BBB opening using focused ultrasound.
- 5a to 5d show the results of confirming the expression levels of Tgfbi, Cst7, Angptl4, and Srgn in the blood of BBB animal models, respectively.
- the present invention can provide a biomarker composition for diagnosing brain diseases caused by brain microvascular damage, including any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn.
- the blood-brain barrier is a barrier that exists between the brain and blood vessels, preventing foreign substances from entering the brain.
- Recent studies have shown that blood-brain barrier leakage is an independent risk factor. Therefore, in order to study brain diseases caused by brain microvascular damage, such as dementia and Parkinson's disease, blood-brain barrier damaged animal models are needed.
- brain diseases caused by brain microvascular damage such as dementia and Parkinson's disease
- blood-brain barrier damaged animal models are needed.
- real-time observation is difficult and it is difficult to distinguish the functions of individual cellular elements. It is necessary. Accordingly, the present inventors irradiated mice with focused ultrasound to create a blood-brain barrier (BBB) patency model.
- BBB blood-brain barrier
- the degree of patency of the blood-brain barrier was controlled by adjusting the intensity of specific focused ultrasound, and optimal damage and mild damage were caused by the control, thereby creating an optimal BBB degeneration mouse model and a mild BBB degeneration mouse model.
- a biological analysis method transciptome sequencing analysis, was performed to discover diagnostic markers for brain diseases caused by brain microvascular damage.
- RNA sequencing analysis under the mild damage BBB condition where BBB damage can be expected to be moderate, about 518 genes were increased and 174 genes were decreased at 48 hours showed a tendency to become (Fig. 3). Based on the above results, blood biomarker candidates under mild damage BBB conditions were selected.
- Tgfbi, Cst7, Angptl4 and Srgn 4 genes encoding water-soluble proteins detectable in blood and increased by more than 3 times compared to the control were selected as candidates.
- Tgfbi, Cst7, Angptl4 and Srgn as a result of confirming in the blood of an optimal BBB degenerated mouse model and a mild BBB degenerated mouse model, the expression of Tgfbi, Cst7, Angptl4 and Srgn was increased over time after BBB opening under optimal and mild damaged conditions. It can be seen that this increases gradually over time. Through this, the possibility of discovering blood biomarkers for brain diseases caused by brain microvascular damage was confirmed (FIGS. 5a to 5d).
- TGFBI Transforming growth factor beta induced
- NM_000358.3 Human
- NM_009369.5 Mae
- Cst7 (Cystatin-F) is known to inhibit papain and cathepsin L, and is known to be involved in immune regulation by inhibiting targets in the hematopoietic system. There is no known correlation between brain diseases caused by cerebral microvascular damage. Information on Cst7 of the present invention is preferably disclosed in, for example, NCBI gene ID NM_003650.4 (Mouse, Human), but is not limited thereto.
- Angptl4 (Angiopoietin like 4) is induced in hypoxic conditions in various cells and is a target of Peroxisome proliferator-activated receptor.
- Angptl4 information of the present invention is preferably disclosed in, for example, NCBI ID NM_001039667.3 (Human), NM_016109, NM_139314.3 (Human) or NM_020581.2 (Mouse), but is not limited thereto.
- Srgn is also known as hematopoietic proteoglycan core protein or secretory granule proteoglycan core protein, which is known as an intracellular proteoglycan mainly expressed in hematopoietic cells and endothelial cells.
- Serglycin is also known as hematopoietic proteoglycan core protein or secretory granule proteoglycan core protein, which is known as an intracellular proteoglycan mainly expressed in hematopoietic cells and endothelial cells.
- Srgn information of the present invention is disclosed in, for example, NCBI gene ID NM_002727.4 (Human), NM_001321053.2 (Human), NM_001321054.1 (Human), NM_001358965.1 (Mouse) or NM_011157.3 (Mouse) Preferred, but not limited thereto.
- 'diagnosis' means confirming the presence or characteristics of a pathological condition.
- diagnosis is to determine whether or not a brain disease is caused by damage to brain microvessels.
- Brain diseases caused by damage to the cerebral microvessels may preferably include dementia, Alzheimer's, Parkinson's, mild cognitive impairment, and mild traumatic brain injury (MTBI), but are not limited thereto.
- MTBI mild traumatic brain injury
- the brain microvascular damage may be caused by the opening or leakage of the blood-brain barrier.
- a 'biomarker' is a substance capable of diagnosing brain diseases caused by cerebral microvascular damage, that is, a substance capable of diagnosing brain diseases caused by cerebral microvascular damage by distinguishing whether or not they occur in a biological sample, Organic organisms such as polypeptides, nucleic acids (e.g., mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.) that show significant differences between normal individuals and individuals with brain diseases caused by brain microvascular damage molecules, etc.
- nucleic acids e.g., mRNA, etc.
- lipids lipids
- glycolipids glycoproteins
- sugars monosaccharides, disaccharides, oligosaccharides, etc.
- the biomarker consists of Tgfbi, Cst7, Angptl4 and Srgn, as any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn increase over time in a mouse model with mild BBB degeneration.
- the increased expression level of any one or more genes selected from the group supports the development of brain diseases due to cerebral microvascular damage.
- the biomarker is any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn, and the group consisting of Tgfbi, Cst7, Angptl4 and Srgn whose expression is increased over time in animal models with mild BBB degeneration.
- the increased expression level of any one or more genes selected from supports the development of brain diseases due to damage to brain microvessels.
- the present invention can provide a composition for diagnosing brain diseases caused by brain microvascular damage, comprising an agent for measuring the mRNA expression of one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn or the protein activity level thereof. there is.
- any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4, and Srgn of the present invention are significantly increased in a brain disease model caused by brain microvascular damage compared to healthy individuals.
- it is possible to diagnose a degenerative brain disease by measuring its gene mRNA expression or its protein level.
- the measurement of the mRNA expression level of any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn is the presence of biomarker genes in biological samples for the diagnosis of brain diseases caused by brain microvascular damage.
- the amount of mRNA is measured using the preparation used in the method of measuring the level of mRNA transcribed from the target gene.
- the agent for measuring the mRNA level of any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn is preferably an antisense oligonucleotide, primer pair or probe, and Tgfbi, Cst7, Angptl4 and Srgn Since the base sequences of one or more genes selected from the group consisting of are registered in the gene bank, those skilled in the art can design antisense oligonucleotides, primer pairs or probes that specifically amplify specific regions of these genes based on the sequences. there is.
- measuring the protein activity level of any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn is the presence or absence of biomarker proteins in biological samples for the diagnosis of brain diseases caused by damage to brain microvessels.
- the amount of the protein can be confirmed using an antibody that specifically binds to the protein of any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn.
- the antibody is a term known in the art and refers to a specific protein molecule directed against an antigenic site.
- the antibody refers to an antibody that specifically binds to Tgfbi, Cst7, Angptl4 or Srgn, ,
- These antibodies can be prepared by cloning each gene into an expression vector according to a conventional method to obtain a protein encoded by the gene, and from the obtained protein by a conventional method. This includes partial peptides that can be made from the protein.
- the form of the antibody of the present invention is not particularly limited, and a polyclonal antibody, a monoclonal antibody, or any antibody having antigen-binding properties is also included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies.
- the present invention can provide a kit for diagnosing brain diseases caused by brain microvascular damage, including the above-described composition.
- the kit of the present invention may include, in addition to agents for measuring the mRNA expression of Tgfbi, Cst7, Angptl4 or Srgn genes or the protein activity level thereof, one or more other component compositions, solutions or devices suitable for analysis methods, and may be taken in any form.
- agents for measuring the mRNA expression of Tgfbi, Cst7, Angptl4 or Srgn genes or the protein activity level thereof one or more other component compositions, solutions or devices suitable for analysis methods, and may be taken in any form.
- the scope of the present invention is not limited.
- the kit for measuring the mRNA expression level of the Tgfbi, Cst7, Angptl4 or Srgn gene in the present invention may be a kit including essential elements necessary for performing RT-PCR.
- the RT-PCR kit contains, in addition to primer pairs specific for the marker gene, a test tube or other suitable container, reaction buffer, deoxynucleotides (usen), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC- It may include water (DEPC-water), sterile water, and the like.
- the kit of the present invention may be a kit including essential elements required to perform a microarray chip.
- the microarray chip kit includes a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof. It can be easily prepared by a manufacturing method commonly used in the art.
- a micropipetting method using a piezoelectric method or a pin type is used to immobilize the searched marker as a probe DNA molecule on a substrate of a DNA chip. It is preferable to use a method using a spotter of, but is not limited thereto.
- the substrate of the microarray chip is preferably coated with an active group selected from the group consisting of amino-silane, poly-L-lysine, and aldehyde, but is not limited thereto.
- the substrate is preferably selected from the group consisting of slide glass, plastic, metal, silicon, nylon membrane and nitrocellulose membrane, but is not limited thereto.
- the kit for measuring the activity level of the Tgfbi, Cst7, Angptl4 or Srgn protein includes a substrate, an appropriate buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, and a chromogenic substrate for immunological detection of the antibody. etc. may be included.
- a nitrocellulose membrane, a 96-well plate synthesized with polyvinyl resin, a 96-well plate synthesized with polystyrene resin, and glass slide glass may be used as the substrate, and the coloring enzyme may be peroxidase, alkaline phosphatase Alkaline phosphatase, etc.
- FITC FITC
- RITC RITC
- ABTS 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sul phonic acid)
- OPD o-phenylenediamine
- TMB tetramethyl benzidine
- the present invention also relates to the diagnosis of brain diseases caused by damage to brain microvessels, which includes measuring the mRNA expression or protein activity level of any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn from a biological sample. Information provision methods can be provided.
- the method for providing information for diagnosis is a preliminary step for diagnosis, which provides objective basic information necessary for the diagnosis of brain diseases caused by damage to brain microvessels, and the clinical judgment or opinion of a doctor is excluded. .
- 'biological sample' refers to a direct target for measuring the expression level of a target gene or protein separated from an individual, and includes samples such as tissues, cells, whole blood, serum, plasma, saliva or urine .
- the sample of the present invention is a sample for diagnosing the onset of a brain disease in a subject suspected of having a brain disease due to brain microvascular damage, and may preferably be blood.
- the subject is applicable to any mammal, and the mammal includes humans and primates as well as livestock such as cattle, pigs, sheep, horses, dogs, and cats.
- the 'analysis method for measuring mRNA expression level' includes polymerase reaction (PCR), reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase Reaction (Realtime RT-PCR), RNase protection assay (RPA; RNase protection assay), northern blotting, or DNA microarray analysis, but is not limited thereto.
- PCR polymerase reaction
- RT-PCR reverse transcription polymerase reaction
- Competitive RT-PCR competitive reverse transcription polymerase reaction
- Realtime RT-PCR real-time reverse transcription polymerase Reaction
- RNase protection assay RNase protection assay
- northern blotting or DNA microarray analysis
- the 'analysis method for measuring the protein activity level' includes western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay, radioimmunodiffusion, and Oktero Ouchterlony immunodiffusion assay, Rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complete fixation assay, Fluorescence Activated Cell Sorter (FACS) or protein chip ( protein chip) analysis method, etc., but is not limited thereto.
- ELISA enzyme linked immunosorbent assay
- radioimmunoassay radioimmunodiffusion
- Oktero Ouchterlony immunodiffusion assay Rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complete fixation assay, Fluorescence Activated Cell Sorter (FACS) or protein chip ( protein chip) analysis method, etc., but is not limited thereto.
- the method of the present invention may include comparing the measured level of mRNA expression or protein activity of the Tgfbi, Cst7, Angptl4 or Srgn gene with the level measured in a control group.
- the control group was a healthy person who did not develop a brain disease due to brain microvascular damage. Accordingly, in the method of the present invention, when the mRNA expression of the Tgfbi, Cst7, Angptl4 or Srgn gene or the protein activity level thereof is higher than that of the control group, it can be determined that a brain disease caused by damage to brain microvessels is in progress or has already occurred. .
- screening relates to an operation of selecting a substance having a therapeutic effect on brain diseases caused by damage to brain microvessels.
- the candidate substance in step (a) means a substance to be tested for therapeutic activity of brain diseases, such as extracts, proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides, and any molecule of a wide range of compounds.
- brain diseases such as extracts, proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides, and any molecule of a wide range of compounds.
- candidate materials include natural materials as well as synthetic materials.
- the step of measuring the mRNA expression or protein activity level of the Tgfbi, Cst7, Angptl4 or Srgn gene in step (b) is a nucleic acid encoding Tgfbi, Cst7, Angptl4 or Srgn by treatment with a candidate substance in the sample. Alternatively, it is a process of confirming the presence and expression level of Tgfbi, Cst7, Angptl4 or Srgn protein.
- the screening method of the present invention (c) the activity of the mRNA or protein of the Tgfbi, Cst7, Angptl4 or Srgn gene measured in step (b) is the Tgfbi, Cst7, Angptl4 or Srgn gene of the control group not treated with the candidate substance.
- a step of determining the candidate substance as a therapeutic agent for brain disease may be further included.
- the mRNA expression of the Tgfbi, Cst7, Angptl4 or Srgn gene or the activity of the protein thereof by the treatment of the candidate substance is lower than the mRNA expression of the Tgfbi, Cst7, Angptl4 or Srgn gene or the activity of the protein of the candidate substance untreated group.
- the candidate substance can be judged as a brain disease treatment, and on the contrary, the mRNA expression of Tgfbi, Cst7, Angptl4 or Srgn gene or the activity of its protein is the mRNA of Tgfbi, Cst7, Angptl4 or Srgn gene in the candidate substance untreated group. If the expression or its protein activity is similar to or higher than that, it can be determined that it cannot be used as a treatment for brain diseases caused by damage to brain microvessels.
- a method for producing an optimal BBB degenerated mouse model comprising the step of treating the mouse with focused ultrasound at 0.25 to 0.27 MPa for 10 to 230 seconds may be provided.
- the present invention can provide a method for producing a mouse model with mild BBB degeneration, comprising the steps of (a) treating the mouse with focused ultrasound at 0.42 to 0.44 MPa for 10 to 230 seconds.
- Redundant content is omitted in consideration of the complexity of the present specification, and terms not defined otherwise in the present specification have meanings commonly used in the field to which the present invention belongs.
- the system consists of an ultrasonic control system and an ultrasonic irradiation system (compatible with MR environment).
- the ultrasonic control system consists of a function generator and an RF amplifier that can deliver RF power to the ultrasonic transducer, and a power meter that can measure the power delivered to the ultrasonic transducer in real time.
- the ultrasonic irradiation system is composed of an ultrasonic transducer, a 3-axis controller that can precisely control the ultrasonic transducer in three dimensions, and a water tank, and is designed to be operable within the MRI equipment environment.
- the MR image-guided focused ultrasound system is linked with the Bruker 9.4T MRI (BioSpec 94/20 USR - 9.4T, Bruker, MA, USA) system.
- microbubbles DEFINITY®
- a contrast agent for ultrasound imaging are diluted in physiological saline at a ratio of 1:50 and injected into the vein of the animal at a rate of 0.012 ml/s.
- focused ultrasound with a burst length of 10 ms per 1 s was irradiated to the desired brain region based on MR images for 2 minutes.
- MR imaging it is possible to confirm opening and closing of the BBB by comparing images before and after MR contrast agent injection.
- MR images after opening the BBB, a contrast agent was administered, the degree of enhancement over time was confirmed, and images were acquired in the coronal, axial, and sagittal directions.
- Experiments were conducted under optimal ( ⁇ 50%) and mild ( ⁇ 100%) BBB conditions through intensity measurement according to the degree of penetration of the MR contrast agent.
- BBB-modified brain tissue was obtained over time (1 hour, 6 hours, 12 hours, 24 hours, 48 hours), and blood samples were collected from each group of mice. About 1 ml was collected and serum was separated.
- RNA was isolated using an RNA isolation kit (Qiagen). . Total RNA was treated with Dnase to prevent DNA contamination, and random fragmentation was produced for library production. The RNA fragment was reacted with reverse transcriptase to prepare cDNA, and adpators were attached to both ends of the cDNA to perform sequencing. The quality of raw data obtained through sequencing was analyzed, and the expression level was extracted as FPKM (Fragments per kilobase of transcript per milion mapped reads) value through transcript quantification of each sample (FIG. 3).
- FPKM Frragments per kilobase of transcript per milion mapped reads
- Example 3 In order to confirm that the sequencing results of the candidate genes in Example 3 were significant, the expression of the candidate genes was checked using real-time PCR testing technique to confirm the correlation.
- the tissue of the optimal damage BBB animal model and the mild damage BBB animal model prepared by the method of Example 1 were rapidly frozen in liquid nitrogen, and RNA was isolated using the Quiagne RNA isolation kit (Quiagen). did Total RNA was treated with Dnase to prevent DNA contamination, and random fragmentation was produced for library production. RNA fragments were reacted with reverse transcriptase to prepare cDNA.
- qRT-q using the SYBR® Green real-time PCR kit in a 20 ⁇ L reaction volume containing 10 ⁇ L of SYBR® Green master PCR mix, 5 pmol each of forward and reverse primers, 1 ⁇ L of diluted cDNA template, and an appropriate amount of sterile distilled water.
- PCR was performed. Gene amplification conditions were as follows. initial denaturation at 95 °C for 3 min; 40 cycles of denaturation at 95°C for 15 seconds, annealing at 60°C for 60 seconds, and elongation at 72°C for 60 seconds; and a final extension at 72° C. for 5 minutes.
- qRT-PCR was performed on an ABI Step One Plus real-time PCR system. All quantifications were performed with U6 as an internal standard. PCR primer sequences are shown in Table 1 below. As a result, it was confirmed that the gene sequencing results and the qRT-PCR results according to time were similar (FIG. 4).
- Biotin-labeled antibody solution was dispensed by 100ul, incubated at 37 °C for 1 hour, and washed three times with wash buffer for 5 minutes each. After dispensing 100ul of ABC solution, incubating at 37 °C for 30 minutes, washing with wash buffer, adding 90ul of TMB substrate solution, incubating at 37 °C for 30 minutes, adding 100ul of stop solution and using a plate reader (Tecan) The absorbance was measured at a wavelength of 450 nm and the results were analyzed. The expression of Tgfbi in blood was more than doubled at 48 hours at 0.42 MPa.
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Abstract
The present invention relates to a bio-marker composition for diagnosing a brain disease due to brain microvascular damage and, more particularly, to a method for manufacturing an animal model with a brain disease due to brain microvascular damage by precisely classifying local areas of a brain on the basis of an MRI image and inducing blood brain barrier degeneration only in a corresponding local area, a method for screening a bio-marker for diagnosing a brain disease due to brain microvascular damage caused by blood brain barrier degeneration, using the animal model, and a bio-marker composition for diagnosing a brain disease due to brain microvascular damage caused by blood brain barrier degeneration.
Description
본 발명은 뇌미세혈관 손상 뇌질환 진단용 바이오 마커 조성물, 보다 상세하게는, MRI 영상 기반으로 뇌 국소영역을 정밀하게 구분하여 해당 국소 영역에서만 혈뇌장벽 변성을 유도하여, 뇌미세혈관 손상으로 인한 뇌질환 동물 모델을 제조하는 방법, 상기 동물 모델을 이용한 혈뇌장벽 변성으로 인한 뇌미세혈관 손상 뇌질환 진단용 바이오 마커를 스크리닝 하는 방법 및 혈뇌장벽 변성으로 인한 뇌미세혈관 손상 뇌질환 진단용 바이오 마커 조성물에 관한 것이다.The present invention is a biomarker composition for diagnosing brain diseases caused by cerebral microvascular damage, and more specifically, brain diseases caused by cerebral microvascular damage by precisely classifying local areas of the brain based on MRI images and inducing degeneration of the blood-brain barrier only in those local areas. A method for preparing an animal model, a method for screening a biomarker for diagnosing brain microvascular damage encephalopathy due to blood-brain barrier degeneration using the animal model, and a biomarker composition for diagnosing brain microvascular damage encephalopathy due to blood-brain barrier degeneration using the animal model.
혈뇌장벽은 뇌와 혈관 사이에 존재하는 장벽으로, 뇌에 외부 물질이 들어오는 것을 막는다. 혈뇌장벽은 뇌에 외부 물질이 들어오는 것을 막고, 대사에 필요한 물질을 받아들여 뇌를 보호하는 역할을 한다. 예를 들어 뇌에서 세균 또는 바이러스로의 침입을 막고, 포도당 공급과 산소-이산화탄소 교환을 원활하게 한다. 혈액은 혈뇌장벽을 통해 여과되며, 결과적으로 뇌에 다다르는 물질은 물이나 기체 분자, 포도당, 특정 지용성 등 극소수이다. 혈뇌장벽은 혈관을 뇌의 내피세포로 감싸는 모양을 띄며, 내피세포는 서로 밀착 연접(tight junction)되어 있기에 세포 사이로 물질이 지나가는 것이 불가능하다. 이 때문에 혈뇌장벽은 진단을 위한 다양한 영상제뿐 아니라, 종양, 알츠하이머, 파킨슨병 등 뇌에 질병이 생겼을 때 약물이 통과하는 것을 막아 뇌 관련 진단 및 치료 기술 개발에 저해가 되고 있다. The blood-brain barrier is a barrier that exists between the brain and blood vessels, preventing foreign substances from entering the brain. The blood-brain barrier prevents foreign substances from entering the brain and protects the brain by accepting substances necessary for metabolism. For example, it prevents the invasion of bacteria or viruses in the brain, and facilitates glucose supply and oxygen-carbon dioxide exchange. Blood is filtered through the blood-brain barrier, and as a result, very few substances reach the brain, such as water or gas molecules, glucose, and certain fat-soluble substances. The blood-brain barrier has a shape that surrounds blood vessels with brain endothelial cells, and it is impossible for substances to pass between cells because the endothelial cells are in tight junctions with each other. For this reason, the blood-brain barrier prevents the passage of not only various imaging agents for diagnosis, but also drugs when diseases occur in the brain, such as tumors, Alzheimer's disease, and Parkinson's disease, thereby hindering the development of brain-related diagnosis and treatment technologies.
최근 연구에서, 알츠하이머 치매의 최초 신호는 혈뇌장벽의 누출이라는 결과가 나왔다. 미국 서던 캘리포니아대학 의대 신경유전자 연구소(Neurogenetic Institute) 소장 베리슬라프 즐로코비치 박사 연구팀에서는 치매는 주범으로 알려진 뇌 신경세포의 독성 단백질 베타 아밀로이드의 응집(plaque)과 타우 단백질의 엉킴(tangle)과 관계없이 혈뇌장벽 누출이 독립적인 위험요인이라는 연구결과를 발표하였다. 인지기능이 정상인 노인 161명을 대상으로 5년에 걸쳐 각종 테스트를 통해 인지기능을 평가하면서 뇌 영상 검사와 뇌 척수액 분석을 통해 혈뇌장벽의 투과성을 측정한 결과, 인지기능 저하와 혈뇌장벽 누출 사이에 밀접한 연관이 있었다. 특히 기억력이 많이 떨어지는 사람일수록 말초 혈관의 혈뇌장벽 누출이 가장 심한 것으로 나타났다. 치매뿐만 아니라 파킨슨 병 등 퇴행성 뇌질환과 혈뇌장벽 손상에 대한 연관성 연구도 많이 진행 중이다. 상기 연구를 위해 치매, 파킨슨병과 같이 퇴행성 뇌질환을 연구하기 위한, 혈뇌장벽 손상으로 인한 뇌미세혈관 손상으로 인한 뇌질환 동물모델이 필요하다. 그러나 기존에 뇌혈관장벽에 관한 모델로 널리 사용되는 동물 모델의 경우 실시간 관찰이 어려우며 개별 세포요소의 기능을 구별하기 어렵고, 동물실험에 대한 윤리적인 문제가 지속적으로 제기됨에 따라 대안적인 동물 연구 모델이 필요한 실정이다. A recent study found that the first sign of Alzheimer's disease is a leak in the blood-brain barrier. Dr. Berislav Zlokovic's research team, director of the Neurogenetic Institute, School of Medicine, University of Southern California, USA, found that dementia is related to the plaque of beta-amyloid, a toxic protein in brain neurons known as the main culprit, and the tangle of tau protein. reported that blood-brain barrier leakage was an independent risk factor. 161 elderly people with normal cognitive function were evaluated for cognitive function through various tests over 5 years, and blood-brain barrier permeability was measured through brain imaging tests and cerebrospinal fluid analysis. There was a close connection. In particular, the blood-brain barrier leakage of peripheral blood vessels was found to be the most severe in people with poor memory. In addition to dementia, many studies on the relationship between degenerative brain diseases such as Parkinson's disease and damage to the blood-brain barrier are also underway. For the above research, an animal model of brain disease caused by cerebral microvascular damage due to damage to the blood-brain barrier is needed to study degenerative brain diseases such as dementia and Parkinson's disease. However, in the case of animal models widely used as models for the blood-brain barrier, real-time observation is difficult and it is difficult to distinguish the functions of individual cellular elements. It is necessary.
이에 본 발명자들은 혈뇌장벽의 손상 동물 모델을 개발하기 위해 연구를 거듭한 결과, 마우스에 초음파 에너지를 조사하여 혈뇌장벽의 변성을 유도하여 뇌미세혈관 손상으로 인한 뇌 질환 마우스 모델을 제작하였고, 상기 마우스 모델의 뇌 조직에서 분리한 단백질을 분석하여 혈뇌장벽의 변성으로 인한 뇌 미세혈관 손상 뇌질환과 관련된 바이오 마커를 스크리닝한 결과, Tgfbi, Cst7, Angptl4 또는 Srgn이 뇌 미세혈관 손상으로 인한 뇌질환과 관련된 바이오 마커임을 확인함으로써, 본 발명을 완성하였다.As a result of repeated studies to develop an animal model with blood-brain barrier damage, the inventors of the present invention irradiated ultrasonic energy to mice to induce degeneration of the blood-brain barrier to produce a mouse model of brain disease caused by brain microvascular damage, and the mouse Proteins isolated from the brain tissue of the model were analyzed to screen for biomarkers related to brain microvascular damage and encephalopathy due to blood-brain barrier degeneration. By confirming that it is a biomarker, the present invention was completed.
본 발명의 목적은 Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군으로부터 선택되는 어느 하나 이상의 유전자를 포함하는, 뇌 미세혈관 손상으로 인한 뇌 질환 진단용 바이오 마커 조성물을 제공하는데 있다. An object of the present invention is to provide a biomarker composition for diagnosing brain diseases caused by brain microvascular damage, including any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn.
본 발명의 다른 목적은 Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군으로부터 선택되는 어느 하나 이상의 유전자의 mRNA 발현 또는 이의 단백질 활성 수준을 측정하는 제제를 포함하는 뇌 미세혈관 손상으로 인한 뇌 질환 진단용 조성물을 제공하는 것이다. Another object of the present invention is to provide a composition for diagnosing brain diseases caused by cerebral microvascular damage, comprising an agent for measuring the mRNA expression or protein activity level of any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn. is to do
본 발명의 또 다른 목적은 상기 서술한 조성물을 포함하는 뇌 미세혈관 손상으로 인한 뇌 질환 진단용 키트를 제공하는 것이다. Another object of the present invention is to provide a kit for diagnosing brain diseases caused by brain microvascular damage comprising the above-described composition.
본 발명의 또 다른 목적은 하기의 단계를 포함하는 뇌 미세혈관 손상으로 인한 뇌 질환의 예방 또는 치료용 약학적 조성물을 스크리닝 하는 방법을 제공하는데 있다. (a) 생물학적 샘플에 임의의 화합물을 처리하는 단계; (b) 상기 생물학적 샘플로부터 Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군으로부터 선택되는 어느 하나 이상의 유전자 또는 이의 단백질의 발현 수준 또는 활성 수준을 확인하는 단계; 및 (c) 상기 발현 수준을 임의의 화합물을 처리하지 않은 정상 대조군에서의 상기 유전자 또는 이의 발현 단백질의 수준과 또는 활성 수준을 비교하는 단계.Another object of the present invention is to provide a method for screening a pharmaceutical composition for preventing or treating brain diseases caused by brain microvascular damage, comprising the following steps. (a) treating the biological sample with any compound; (b) confirming the expression level or activity level of any one or more genes or proteins thereof selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn from the biological sample; and (c) comparing the expression level with the level of the gene or its expressed protein or activity level in a normal control group not treated with any compound.
본 발명의 다른 목적은 (a) 마우스에 집속 초음파를 0.25 내지 0.27 MPa로 10초 내지 230초 동안 처리는 단계를 포함하는 optimal BBB 변성 마우스 모델을 제작하는 방법을 제공하는데 있다.Another object of the present invention is to provide a method for producing an optimal BBB degenerated mouse model comprising the step of (a) treating the mouse with focused ultrasound at 0.25 to 0.27 MPa for 10 to 230 seconds.
본 발명의 다른 목적은 (a) 마우스에 집속 초음파 0.42 내지 0.44 MPa로 10초 내지 230초 동안 처리하는 단계를 포함하는 mild damaged BBB 변성 마우스 모델을 제작하는 방법을 제공하는데 있다.Another object of the present invention is to provide a method for producing a mildly damaged BBB degenerated mouse model comprising the step of (a) treating the mouse with focused ultrasound at 0.42 to 0.44 MPa for 10 to 230 seconds.
상기 목적을 달성하기 위해, 본 발명은 Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군으로부터 선택되는 어느 하나 이상의 유전자를 포함하는 뇌미세혈관 손상으로 인한 뇌 미세혈관 손상으로 인한 뇌 질환 진단용 바이오 마커 조성물을 제공할 수 있다. In order to achieve the above object, the present invention provides a biomarker composition for diagnosing brain diseases caused by cerebral microvascular damage, including any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn. can do.
본 발명은 또한 Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군으로부터 선택되는 어느 하나 이상의 유전자의 mRNA 발현 또는 이의 단백질 활성 수준을 측정하는 제제를 포함하는 뇌 미세혈관 손상으로 인한 뇌 질환 진단용 조성물을 제공할 수 있다. The present invention can also provide a composition for diagnosing brain diseases caused by cerebral microvascular damage comprising an agent for measuring the mRNA expression or protein activity level of any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn. there is.
상기 제제는 Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군으로부터 선택되는 어느 하나 이상의 유전자에 특이적으로 결합하는 프라이머 또는 프로브일 수 있다. The agent may be a primer or probe that specifically binds to any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn.
상기 제제는 Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군으로부터 선택되는 어느 하나 이상의 유전자의 단백질에 특이적으로 결합하는 항체일 수 있다. The agent may be an antibody that specifically binds to a protein of one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn.
본 발명은 또한 상기 서술한 조성물을 포함하는 뇌 미세혈관 손상으로 인한 뇌 질환 진단용 키트를 제공할 수 있다. The present invention can also provide a kit for diagnosing brain diseases caused by brain microvascular damage comprising the above-described composition.
본 발명은 또한, 뇌미세혈관의 손상으로 인한 뇌 질환을 진단하기위한 Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군에서 선택된 적어도 어느 하나의 유전자를 포함하는 바이오 마커 조성물의 용도와 생물학적 시료로부터 Tgfbi, Cst7, Angptl4 또는 Srgn 유전자의 mRNA 발현 수준 또는 이의 단백질 활성 수준을 측정하는 단계;를 포함하는 뇌미세혈관의 손상으로 인한 뇌 질환의 진단 방법을 제공할 수 있다.The present invention also relates to the use of a biomarker composition comprising at least one gene selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn for diagnosing brain diseases caused by brain microvascular damage and Tgfbi, Cst7 from biological samples. It is possible to provide a method for diagnosing brain diseases caused by damage to brain microvessels including; measuring the mRNA expression level or the protein activity level of the Angptl4 or Srgn gene.
본 발명은 또한 생물학적 시료로부터 Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군으로부터 선택되는 어느 하나 이상의 유전자의 mRNA 발현 또는 이의 단백질 활성 수준을 측정하는 단계를 포함하는 뇌 미세혈관 손상으로 인한 뇌 질환 진단을 위한 정보제공방법을 제공할 수 있다. The present invention is also directed to a method for diagnosing brain diseases caused by brain microvascular damage comprising measuring the mRNA expression or protein activity level of any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn from a biological sample. How to provide information can be provided.
본 발명은 하기의 단계를 포함하는 뇌 미세혈관 손상으로 인한 뇌 질환의 예방 또는 치료용 약학적 조성물을 스크리닝 하는 방법을 제공할 수 있다: (a) 생물학적 샘플에 임의의 화합물을 처리하는 단계; (b) 상기 생물학적 샘플로부터 Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군으로부터 선택되는 어느 하나 이상의 유전자 또는 이의 단백질의 발현 수준 또는 활성 수준을 확인하는 단계; 및 (c) 상기 발현 수준을 임의의 화합물을 처리하지 않은 정상대조군에서의 상기 유전자 또는 이의 발현 단백질의 수준과 또는 활성 수준을 비교하는 단계.The present invention may provide a method for screening a pharmaceutical composition for preventing or treating brain diseases caused by brain microvascular damage, comprising the following steps: (a) treating a biological sample with an arbitrary compound; (b) confirming the expression level or activity level of any one or more genes or proteins thereof selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn from the biological sample; and (c) comparing the expression level with the level of the gene or its expressed protein or activity level in a normal control group not treated with any compound.
본 발명은 (a) 마우스에 집속 초음파를 0.25 내지 0.27 MPa로 10초 내지 230초 동안 처리는 단계를 포함하는 optimal BBB 변성 마우스 모델을 제작하는 방법을 제공할 수 있다. The present invention may provide a method for producing an optimal BBB degenerated mouse model comprising the step of (a) treating the mouse with focused ultrasound at 0.25 to 0.27 MPa for 10 to 230 seconds.
본 발명은 (a) 마우스에 집속 초음파 0.42 내지 0.44 MPa로 10초 내지 230초 동안 처리하는 단계를 포함하는 mild damaged BBB 변성 마우스 모델을 제작하는 방법을 제공할 수 있다.The present invention may provide a method for producing a mildly damaged BBB degenerated mouse model comprising the step of (a) treating the mouse with focused ultrasound at 0.42 to 0.44 MPa for 10 seconds to 230 seconds.
본 발명은 집속 초음파를 마우스에 조사하여 혈뇌장벽(blood-brain barrier; BBB)의 개통으로 인한 뇌 미세혈관 손상 뇌 질환 마우스 모델을 제작하였다. 또한 상기 집속 초음파의 세기를 조절하여 혈뇌장벽의 개통 정도를 조절 하였고, 상기 조절로 경도 손상(optimal damage) 및 중등 손상(mild damage)를 일으켜, optimal BBB 변성 마우스 모델 및 mild BBB 변성 마우스 모델을 제작 하였다. 상기 mild BBB 변성 마우스의 뇌를 가지고, 생물학적 분석 방법인 transcriptome sequencing analysis를 진행하여, BBB의 변성으로 인한 뇌질환 진단 마커를 발굴한 결과 Tgfbi, Cst7, Angptl4 또는 Srgn이 뇌 미세혈관 손상으로 인한 뇌 질환을 진단할 수 있다는 것을 확인하였다. 상기 시험결과를 바탕으로 optimal BBB 변성 동물 모델 및 mild BBB 변성 동물 모델의 혈액에서 Tgfbi, Cst7, Angptl4 및 Srgn의 발현 정도를 확인인한 결과 증가하는 것을 확인하였다. 상기의 결과를 토대로 Tgfbi, Cst7, Angptl4 또는 Srgn을 이용하여 뇌 미세혈관 손상으로 인한 뇌 질환, 예를 들어 치매, 뇌암, 파킨슨, 뇌진탕 (TBI) 또는 뇌졸중을 진단 할 수 있는 효과가 있다.In the present invention, a mouse model of brain microvascular damage and brain disease caused by opening of the blood-brain barrier (BBB) was prepared by irradiating a mouse with focused ultrasound. In addition, the degree of patency of the blood-brain barrier was controlled by adjusting the intensity of the focused ultrasound, and the control caused optimal and moderate damage, thereby producing an optimal BBB degenerated mouse model and a mild BBB degenerated mouse model did With the brain of the mild BBB-degenerated mouse, transcriptome sequencing analysis, a biological analysis method, was performed to discover diagnostic markers for brain diseases caused by BBB degeneration. was confirmed to be diagnosable. Based on the above test results, it was confirmed that the expression levels of Tgfbi, Cst7, Angptl4 and Srgn were increased in the blood of optimal BBB degeneration animal models and mild BBB degeneration animal models. Based on the above results, there is an effect of diagnosing brain diseases such as dementia, brain cancer, Parkinson's, concussion (TBI) or stroke caused by brain microvascular damage using Tgfbi, Cst7, Angptl4 or Srgn.
도 1은 집속 초음파를 이용한 BBB 개통 동물모델을 제작하는 방법의 모식도이다.1 is a schematic diagram of a method for manufacturing an animal model of BBB opening using focused ultrasound.
도 2는 자기공명영상장치를 이용하여 집속 초음파를 이용한 BBB 동물모델의 BBB 변성 정도를 확인한 결과이다. 2 is a result of confirming the degree of BBB degeneration in a BBB animal model using focused ultrasound using a magnetic resonance imaging device.
도 3은 유전체 분석을 통한 BBB 변성 뇌 조직에서 유전자 발현의 변화를 확인한 결과이다. 3 is a result of confirming changes in gene expression in BBB degenerated brain tissue through genome analysis.
도 4는 유전체 분석 결과를 바탕으로 혈액 바이오 마커 후보를 선정한 결과이다. 4 is a result of selecting blood biomarker candidates based on the genome analysis results.
도 5a 내지 도 5d는 BBB 동물모델의 혈에서 Tgfbi, Cst7, Angptl4 및 Srgn의 발현 정도를 각각 확인한 결과이다.5a to 5d show the results of confirming the expression levels of Tgfbi, Cst7, Angptl4, and Srgn in the blood of BBB animal models, respectively.
이하, 본 발명을 보다 상세히 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명은 Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군으로부터 선택되는 어느 하나 이상의 유전자를 포함하는 뇌 미세혈관 손상으로 인한 뇌 질환 진단용 바이오 마커 조성물을 제공할 수 있다. The present invention can provide a biomarker composition for diagnosing brain diseases caused by brain microvascular damage, including any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn.
혈뇌장벽은 뇌와 혈관 사이에 존재하는 장벽으로, 뇌에 외부 물질이 들어오는 것을 막는다. 최근 연구에서 혈뇌장벽 누출이 독립적인 위험요인이라는 연구결과가 발표되었다. 따라서 치매, 파킨슨병과 같은 뇌 미세혈관 손상으로 인한 뇌질환을 연구하기 위해, 혈뇌장벽 손상 동물모델이 필요하다. 그러나 기존에 뇌혈관장벽에 관한 모델로 널리 사용되는 동물 모델의 경우 실시간 관찰이 어려우며 개별 세포요소의 기능을 구별하기 어렵고, 동물실험에 대한 윤리적인 문제가 지속적으로 제기됨에 따라 대안적인 동물 연구 모델이 필요한 실정이다. 이에 본 발명자들은 집속 초음파를 마우스에 조사하여 혈뇌장벽(blood-brain barrier; BBB)의 개통 모델을 제작하였다. 특정 집속 초음파의 세기를 조절하여 혈뇌장벽의 개통 정도를 조절 하였고, 상기 조절로 경도 손상(optimal damage) 및 중등 손상(mild damage)을 일으켜, optimal BBB 변성 마우스 모델 및 mild BBB 변성 마우스 모델을 제작하였다. 상기 mild BBB 변성 마우스의 뇌를 가지고, 생물학적 분석 방법인 transciptome sequencing analysis를 진행하여, 뇌 미세혈관 손상으로 인한 뇌질환 진단 마커를 발굴하였다. BBB의 손상이 중반 정도로 예상할 수 있는 mild damage BBB 조건에서 RNA sequencing 분석 결과를 통해 시간에 따른 유전자 발현 변화를 control 그룹과 비교하였을 때, 48시간 째 약 518개의 유전자가 증가되었고 174개의 유전자가 감소되는 경향을 나타내었다 (도 3). 상기의 결과를 바탕으로 mild damage BBB 조건에서의 혈액 바이오 마커 후보군을 선정하였다. 518개의 유전자중 Control 대비 3배 이상 증가되고 혈액에서 검출할 수 있는 수용성의 단백질을 코딩하고 있는 4개(Tgfbi, Cst7, Angptl4 및 Srgn)의 유전자를 후보군으로 선정하였다. 상기 유전자 Tgfbi, Cst7, Angptl4 및 Srgn의 경우에는 optimal BBB 변성 마우스 모델 및 mild BBB 변성 마우스 모델의 혈액에서 확인한 결과, Tgfbi, Cst7, Angptl4 및 Srgn의 발현은 optimal condition과 mild damaged condition에서 BBB 개통 후 시간이 지남에 따라 점차 증가함을 알 수 있다. 이를 통해 뇌미세혈관 손상에 의한 뇌질환의 혈액 바이오 마커 발굴 가능성을 확인하였다 (도 5a 내지 도 5d). The blood-brain barrier is a barrier that exists between the brain and blood vessels, preventing foreign substances from entering the brain. Recent studies have shown that blood-brain barrier leakage is an independent risk factor. Therefore, in order to study brain diseases caused by brain microvascular damage, such as dementia and Parkinson's disease, blood-brain barrier damaged animal models are needed. However, in the case of animal models widely used as models for the blood-brain barrier, real-time observation is difficult and it is difficult to distinguish the functions of individual cellular elements. It is necessary. Accordingly, the present inventors irradiated mice with focused ultrasound to create a blood-brain barrier (BBB) patency model. The degree of patency of the blood-brain barrier was controlled by adjusting the intensity of specific focused ultrasound, and optimal damage and mild damage were caused by the control, thereby creating an optimal BBB degeneration mouse model and a mild BBB degeneration mouse model. . With the brain of the mild BBB-degenerated mouse, a biological analysis method, transciptome sequencing analysis, was performed to discover diagnostic markers for brain diseases caused by brain microvascular damage. When comparing gene expression changes over time with the control group through RNA sequencing analysis under the mild damage BBB condition where BBB damage can be expected to be moderate, about 518 genes were increased and 174 genes were decreased at 48 hours showed a tendency to become (Fig. 3). Based on the above results, blood biomarker candidates under mild damage BBB conditions were selected. Among the 518 genes, 4 genes (Tgfbi, Cst7, Angptl4 and Srgn) encoding water-soluble proteins detectable in blood and increased by more than 3 times compared to the control were selected as candidates. In the case of the above genes Tgfbi, Cst7, Angptl4 and Srgn, as a result of confirming in the blood of an optimal BBB degenerated mouse model and a mild BBB degenerated mouse model, the expression of Tgfbi, Cst7, Angptl4 and Srgn was increased over time after BBB opening under optimal and mild damaged conditions. It can be seen that this increases gradually over time. Through this, the possibility of discovering blood biomarkers for brain diseases caused by brain microvascular damage was confirmed (FIGS. 5a to 5d).
본 발명에 있어서, TGFBI(Transforming growth factor beta induced)는, 단백질은 세포-콜라겐 상호 작용에 역할을 하며 연골 내 골 형성에 관여하는 것으로 알려져있으나, 상기 유전자의 뇌미세혈관 손상으로 인한 뇌 질환의 상관관계에 대해서는 전혀 개시되어 있지않다. 본 발명의 TGFBI의 정보는 예를 들어 NCBI gene ID NM_000358.3(Human), NM_009369.5(Mouse)에 개시된 것이 바람직하나, 이에 제한되는 것은 아니다. In the present invention, TGFBI (Transforming growth factor beta induced) is a protein known to play a role in cell-collagen interactions and to be involved in bone formation in cartilage, but the correlation of brain diseases caused by brain microvascular damage of the gene The relationship is not disclosed at all. Information on TGFBI of the present invention is preferably disclosed in, for example, NCBI gene ID NM_000358.3 (Human), NM_009369.5 (Mouse), but is not limited thereto.
본 발명에 있어서, Cst7(Cystatin-F)은 파파인(papain)과 카텝신 L(cathepsin L)을 억제하는 것으로 알려져있으며, 조혈계에서 표적을 억제하여 면역 조절에 관여하는 것으로 알려져있으나, 상기 유전자의 뇌미세혈관 손상으로 인한 뇌 질환의 상관관계에 대해서는 전혀 알려진 바 없다. 본 발명의 Cst7의 정보는 예를 들어 NCBI gene ID NM_003650.4(Mouse, Human) 에 개시된 것이 바람직하나, 이에 제한되는 것은 아니다.In the present invention, Cst7 (Cystatin-F) is known to inhibit papain and cathepsin L, and is known to be involved in immune regulation by inhibiting targets in the hematopoietic system. There is no known correlation between brain diseases caused by cerebral microvascular damage. Information on Cst7 of the present invention is preferably disclosed in, for example, NCBI gene ID NM_003650.4 (Mouse, Human), but is not limited thereto.
본 발명에 있어서, Angptl4 (Angiopoietin like 4)은 다양한 세포의 저산소 조건에서 유도되며 Peroxisome proliferator-activated receptor의 표적이 된다. 그러나 상기 유전자의 뇌미세혈관 손상으로 인한 뇌 질환의 상관관계에 대해서는 전혀 개시되어 있지 않다. 본 발명의 Angptl4의 정보는 예를 들어 NCBI ID NM_001039667.3(Human), NM_016109, NM_139314.3(Human) 또는 NM_020581.2(Mouse)에 개시된 것이 바람직하나 이에 제한되는 것은 아니다. In the present invention, Angptl4 (Angiopoietin like 4) is induced in hypoxic conditions in various cells and is a target of Peroxisome proliferator-activated receptor. However, the correlation of the gene with brain diseases caused by brain microvascular damage has not been disclosed at all. Angptl4 information of the present invention is preferably disclosed in, for example, NCBI ID NM_001039667.3 (Human), NM_016109, NM_139314.3 (Human) or NM_020581.2 (Mouse), but is not limited thereto.
본 발명에 있어서, Srgn (Serglycin)은 조혈 프로테오글리칸 코어 단백질 또는 분비 과립 프로테오글리칸 코어 단백질로도 알려져 있으며, 이는 주로 조혈 세포와 내피 세포에서 발현되는 세포 내 프로테오글리칸으로 알려져 있다. 그러나 상기 유전자의 뇌미세혈관 손상으로 인한 뇌 질환의 상관관계에 대해서는 전혀 개시되어 있지 않다. 본 발명의 Srgn의 정보는 예를 들어 NCBI gene ID NM_002727.4(Human), NM_001321053.2(Human), NM_001321054.1(Human), NM_001358965.1(Mouse) 또는 NM_011157.3(Mouse) 에 개시된 것이 바람직하나, 이에 제한되는 것은 아니다. In the present invention, Srgn (Serglycin) is also known as hematopoietic proteoglycan core protein or secretory granule proteoglycan core protein, which is known as an intracellular proteoglycan mainly expressed in hematopoietic cells and endothelial cells. However, the correlation of the gene with brain diseases caused by brain microvascular damage has not been disclosed at all. Srgn information of the present invention is disclosed in, for example, NCBI gene ID NM_002727.4 (Human), NM_001321053.2 (Human), NM_001321054.1 (Human), NM_001358965.1 (Mouse) or NM_011157.3 (Mouse) Preferred, but not limited thereto.
본 발명에 있어서, '진단'은 병리 상태의 존재 또는 특징을 확인하는 것을 의미한다. 본 발명의 목적상, 진단이란 뇌 미세혈관의 손상으로 인한 뇌질환의 발병 여부를 확인하는 것이다. 상기 뇌미세혈관의 손상으로 인한 뇌 질환은 바람직하게는 치매, 알츠하이머, 파킨슨, 경도인지장애 (mild cognitive impairment) 및 경도의 외상성뇌질환 (MTBI, mild traumatic brain injury) 이 포함될 수 있으나 이에 제한되지 않는다. 또한 상기 뇌미세혈관 손상은 뇌혈관장벽의 개통 또는 누출로 발생하는 것일 수 있다. In the present invention, 'diagnosis' means confirming the presence or characteristics of a pathological condition. For the purpose of the present invention, diagnosis is to determine whether or not a brain disease is caused by damage to brain microvessels. Brain diseases caused by damage to the cerebral microvessels may preferably include dementia, Alzheimer's, Parkinson's, mild cognitive impairment, and mild traumatic brain injury (MTBI), but are not limited thereto. . In addition, the brain microvascular damage may be caused by the opening or leakage of the blood-brain barrier.
본 발명에 있어서, '바이오 마커'란 뇌미세혈관 손상으로 인한 뇌 질환을 진단할 수 있는 물질, 즉 생물학적 시료에서 뇌미세혈관 손상으로 인한 뇌질환의 발생 여부를 구분하여 진단할 수 있는 물질로, 정상 개체와 뇌미세혈관 손상으로 인한 뇌질환 개체 간의 유의적인 차이를 보이는 폴리펩타이드, 핵산 (예:mRNA 등), 지질, 당지질, 당단백질, 당(단당류, 이당류, 올리고당류 등) 등과 같은 유기 생체 분자 등을 포함한다. 본 발명의 목적상 바이오 마커는 Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군으로부터 선택되는 어느 하나 이상의 유전자가 mild BBB 변성 마우스 모델에서 시간이 경과함에 따라 증가하는 바, Tgfbi, Cst7, Angptl4 및 Srgn로 이루어진 군으로부터 선택되는 어느 하나 이상의 유전자의 증가된 발현 정도가 뇌미세혈관 손상으로 인한 뇌 질환의 발병을 뒷받침한다. In the present invention, a 'biomarker' is a substance capable of diagnosing brain diseases caused by cerebral microvascular damage, that is, a substance capable of diagnosing brain diseases caused by cerebral microvascular damage by distinguishing whether or not they occur in a biological sample, Organic organisms such as polypeptides, nucleic acids (e.g., mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.) that show significant differences between normal individuals and individuals with brain diseases caused by brain microvascular damage molecules, etc. For the purpose of the present invention, the biomarker consists of Tgfbi, Cst7, Angptl4 and Srgn, as any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn increase over time in a mouse model with mild BBB degeneration. The increased expression level of any one or more genes selected from the group supports the development of brain diseases due to cerebral microvascular damage.
본 발명의 목적상 바이오 마커는 Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군으로부터 선택되는 어느 하나 이상의 유전자로, mild BBB 변성 동물모델에서 시간에 따라서 발현이 증가된 Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군으로부터 선택되는 어느 하나 이상의 유전자의 증가된 발현 정도가 뇌미세혈관의 손상으로 인한 뇌 질환의 발병을 뒷받침한다.For the purpose of the present invention, the biomarker is any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn, and the group consisting of Tgfbi, Cst7, Angptl4 and Srgn whose expression is increased over time in animal models with mild BBB degeneration. The increased expression level of any one or more genes selected from supports the development of brain diseases due to damage to brain microvessels.
본 발명은 Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군으로부터 선택되는 어느 하나 이상의 유전자의 mRNA 발현 또는 이의 단백질 활성 수준을 측정하는 제제를 포함하는 뇌미세혈관의 손상으로 인한 뇌 질환 진단용 조성물을 제공할 수 있다. The present invention can provide a composition for diagnosing brain diseases caused by brain microvascular damage, comprising an agent for measuring the mRNA expression of one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn or the protein activity level thereof. there is.
본 발명의 일 실시예에 따르면, 본 발명의 Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군으로부터 선택되는 어느 하나 이상의 유전자는 건강한 정상인에 비하여 뇌미세혈관의 손상으로 인한 뇌 질환 모델에서 유의하게 증가하는 바, 이의 유전자 mRNA 발현 또는 이의 단백질 수준을 측정함으로써 퇴행성 뇌질환의 진단이 가능하다. According to one embodiment of the present invention, any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4, and Srgn of the present invention are significantly increased in a brain disease model caused by brain microvascular damage compared to healthy individuals. However, it is possible to diagnose a degenerative brain disease by measuring its gene mRNA expression or its protein level.
본 발명에 있어서, Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군으로부터 선택되는 어느 하나 이상의 유전자의 mRNA 발현 수준의 측정은 뇌미세혈관의 손상으로 인한 뇌 질환의 진단을 위하여 생물학적 시료에서 바이오 마커 유전자의 존재 여부와 발현 정도를 확인하는 과정으로, 표적 유전자부터 전사된 mRNA의 수준을 측정하는 방법에 사용되는 제제를 이용해 mRNA의 양을 측정한다. 구체적으로 본 발명에서 Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군으로부터 선택되는 어느 하나 이상의 유전자의 mRNA 수준을 측정하는 제제는 바람직하게는 안티센스 올리고뉴클레오티드, 프라이머 쌍 또는 프로브이며, Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군으로부터 선택되는 어느 하나 이상의 유전자의 염기 서열이 유전자 은행에 등록되어 있으므로 당업자는 상기 서열을 바탕으로 이들 유전자의 특정 영역을 특이적으로 증폭하는 안티센스 올리고뉴클레오티드, 프라이머 쌍 또는 프로브를 디자인 할 수 있다. In the present invention, the measurement of the mRNA expression level of any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn is the presence of biomarker genes in biological samples for the diagnosis of brain diseases caused by brain microvascular damage. In the process of confirming the presence and expression level, the amount of mRNA is measured using the preparation used in the method of measuring the level of mRNA transcribed from the target gene. Specifically, in the present invention, the agent for measuring the mRNA level of any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn is preferably an antisense oligonucleotide, primer pair or probe, and Tgfbi, Cst7, Angptl4 and Srgn Since the base sequences of one or more genes selected from the group consisting of are registered in the gene bank, those skilled in the art can design antisense oligonucleotides, primer pairs or probes that specifically amplify specific regions of these genes based on the sequences. there is.
본 발명에 있어서, Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군으로부터 선택되는 어느 하나 이상의 유전자의 단백질 활성 수준 측정은 뇌미세혈관의 손상으로 인한 뇌 질환의 진단을 위하여 생물학적 시료에서 바이오 마커 단백질의 존재 여부와 발현 정도를 확인하는 과정으로, 바람직하게는 Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군으로부터 선택되는 어느 하나 이상의 유전자의 단백질에 대하여 특이적으로 결합하는 항체를 이용해 단백질의 양을 확인할 수 있다. In the present invention, measuring the protein activity level of any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn is the presence or absence of biomarker proteins in biological samples for the diagnosis of brain diseases caused by damage to brain microvessels. As a process for confirming the expression level of and, preferably, the amount of the protein can be confirmed using an antibody that specifically binds to the protein of any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn.
상기 항체는 당해 분야에서 공지된 용어로서 항원성 부위에 대해서 지시되는 특이적인 단백질 분자를 지칭하는 것으로, 본 발명의 목적상 항체는 Tgfbi, Cst7, Angptl4 또는 Srgn에 특이적으로 결합하는 항체를 의미하며, 이러한 항체는 각 유전자를 통상적인 방법에 따라 발현벡터에 클로닝하여 상기 유전자에 의해 코딩되는 단백질을 얻고, 얻어진 단백질로부터 통상적인 방법에 의해 제조될 수 있다. 여기에는 상기 단백질에서 만들어 질 수 있는 부분 펩티드도 포함된다. 본 발명의 항체의 형태는 특별히 제한되지 않으며 폴리클로날 항체, 모노클로날 항체 또는 항원 결합성을 갖는 것이면 그것의 일부도 본 발명의 항체에 포함되고 모든 면역글로불린 항체가 포함된다. 나아가, 본 발명의 항체에는 인간화 항체 등의 특수항체도 포함된다. The antibody is a term known in the art and refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, the antibody refers to an antibody that specifically binds to Tgfbi, Cst7, Angptl4 or Srgn, , These antibodies can be prepared by cloning each gene into an expression vector according to a conventional method to obtain a protein encoded by the gene, and from the obtained protein by a conventional method. This includes partial peptides that can be made from the protein. The form of the antibody of the present invention is not particularly limited, and a polyclonal antibody, a monoclonal antibody, or any antibody having antigen-binding properties is also included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies.
본 발명은 상기 서술한 조성물을 포함하는 뇌미세혈관의 손상으로 인한 뇌 질환 진단용 키트를 제공할 수 있다. The present invention can provide a kit for diagnosing brain diseases caused by brain microvascular damage, including the above-described composition.
본 발명의 키트에는 Tgfbi, Cst7, Angptl4 또는 Srgn 유전자의 mRNA 발현 또는 이의 단백질 활성 수준을 측정하는 제제 외에, 분석방법에 적합한 한 종류 이상의 다른 구성성분 조성물, 용액 또는 장치가 포함될 수 있으며, 어떠한 형태로든 본 발명의 범위를 제한하지 않는다. The kit of the present invention may include, in addition to agents for measuring the mRNA expression of Tgfbi, Cst7, Angptl4 or Srgn genes or the protein activity level thereof, one or more other component compositions, solutions or devices suitable for analysis methods, and may be taken in any form. The scope of the present invention is not limited.
구체적인 일례로서, 본 발명에서 상기 Tgfbi, Cst7, Angptl4 또는 Srgn 유전자의 mRNA 발현 수준을 측정하기 위한 키트는 RT-PCR을 수행하기 위해 필요한 필수 요소를 포함하는 키트일 수 있다. 상기 RT-PCR 키트는 마커 유전자에 대한 특이적인 프라이머 쌍 외에도 테스트 튜브 또는 다른 적절한 컨테이너, 반응 완충액, 데옥시뉴클레오티드(우센), Taq-중합효소 및 역전사효소와 같은 효소, DNase, RNase 억제제, DEPC-물(DEPC-water), 멸균수 등을 포함할 수 있다. As a specific example, the kit for measuring the mRNA expression level of the Tgfbi, Cst7, Angptl4 or Srgn gene in the present invention may be a kit including essential elements necessary for performing RT-PCR. The RT-PCR kit contains, in addition to primer pairs specific for the marker gene, a test tube or other suitable container, reaction buffer, deoxynucleotides (usen), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC- It may include water (DEPC-water), sterile water, and the like.
또한, 본 발명의 키트는 마이크로어레이 칩을 수행하기 위해 필요한 필수 요소를 포함하는 키트일 수 있다. 상기 마이크로어레이 칩 키트는, 유전자 또는 그의 단편에 해당하는 cDNA가 프로브로 부착되어 있는 기판을 포함하고 기판은 정량 대조구 유전자 또는 그의 단편에 해당하는 cDNA를 포함할 수 있으며, 본 발명의 마커를 이용 하여 당업계에서 통상적으로 사용되는 제조 방법에 의하여 용이하게 제조될 수 있다. 마이크로어레이 칩을 제작하기 위해서, 상기 탐색된 마커를 탐침 DNA 분자로 이용하여 DNA 칩의 기판상에 고정화시키기 위해 파이조일렉트릭(piezoelectric) 방식을 이용한 마이크로피펫팅(micropipetting)법 또는 핀(pin) 형태의 스폿터(spotter)를 이용한 방법 등을 사용하는 것이 바람직하나 이에 한정되는 것은 아니다. 상기 마이크로어레이 칩의 기판은 아미노-실란(amino-silane), 폴리-L-라이신(poly-Llysine) 및 알데히드(aldehyde)로 이루어진 군에서 선택되는 활성기가 코팅된 것이 바람직하나, 이에 한정되는 것은 아니다. 또한, 상기 기판은 슬라이드 글래스, 플라스틱, 금속, 실리콘, 나일론 막 및 니트로셀룰로스 막(nitrocellulose membrane)으로 이루어진 군에서 선택되는 것이 바람직하나 이에 한정되는 것은 아니다.In addition, the kit of the present invention may be a kit including essential elements required to perform a microarray chip. The microarray chip kit includes a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof. It can be easily prepared by a manufacturing method commonly used in the art. In order to fabricate a microarray chip, a micropipetting method using a piezoelectric method or a pin type is used to immobilize the searched marker as a probe DNA molecule on a substrate of a DNA chip. It is preferable to use a method using a spotter of, but is not limited thereto. The substrate of the microarray chip is preferably coated with an active group selected from the group consisting of amino-silane, poly-L-lysine, and aldehyde, but is not limited thereto. . In addition, the substrate is preferably selected from the group consisting of slide glass, plastic, metal, silicon, nylon membrane and nitrocellulose membrane, but is not limited thereto.
또한, 본 발명에서 상기 Tgfbi, Cst7, Angptl4 또는 Srgn 단백질 활성 수준을 측정하기 위한 키트는 항체의 면역학적 검출을 위하여 기질, 적당한 완충 용액, 발색 효소 또는 형광 물질로 표지된 2차 항체, 및 발색 기질 등을 포함할 수 있다. 상기에서 기질은 니트로셀룰로오스 막, 폴리비닐 수지로 합성된 96 웰 플레이트, 폴리스틸렌 수지로 합성된 96 웰 플레이트 및 유리로 된 슬라이드 글라스 등이 이용될 수 있고, 발색효소는 퍼옥시다아제(peroxidase), 알칼라인 포스파타아제(alkaline phosphatase) 등이 사용될 수 있고, 형광물질은 FITC, RITC등이 사용될 수 있고, 발색 기질액은 ABTS(2,2'-아지노-비스-(3-에틸벤조티아졸린-6-설폰산)) 또는 OPD(o-페닐렌디아민), TMB(테트라메틸 벤지딘)가 사용될 수 있으나, 이에 한정되는 것은 아니다.In addition, in the present invention, the kit for measuring the activity level of the Tgfbi, Cst7, Angptl4 or Srgn protein includes a substrate, an appropriate buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, and a chromogenic substrate for immunological detection of the antibody. etc. may be included. In the above, a nitrocellulose membrane, a 96-well plate synthesized with polyvinyl resin, a 96-well plate synthesized with polystyrene resin, and glass slide glass may be used as the substrate, and the coloring enzyme may be peroxidase, alkaline phosphatase Alkaline phosphatase, etc. may be used, FITC, RITC, etc. may be used for the fluorescent substance, and ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sul phonic acid)) or OPD (o-phenylenediamine), TMB (tetramethyl benzidine) may be used, but is not limited thereto.
본 발명은 또한 생물학적 시료로부터 Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군으로부터 선택되는 어느 하나 이상의 유전자의 mRNA 발현 또는 이의 단백질 활성 수준을 측정하는 단계를 포함하는 뇌미세혈관의 손상으로 인한 뇌 질환 진단을 위한 정보제공방법을 제공할 수 있다. The present invention also relates to the diagnosis of brain diseases caused by damage to brain microvessels, which includes measuring the mRNA expression or protein activity level of any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn from a biological sample. Information provision methods can be provided.
본 발명의 일 실시예에 따르면, 집속초음파 조사에 따른 BBB 개통후 시간에 따른 생물학적 반응을 확인하기 위해서 시간에 따라 (1시간, 6시간, 12시간, 24시간, 48시간) mild BBB 변성 뇌조직을 획득한 후, RNA sequencing 분석 결과를 통해 시간에 따른 유전자 발현 변화를 control 그룹과 비교하였을 때, 48시간 째 약 518개의 유전자가 증가되었고 174개의 유전자가 감소되는 경향을 나태었고, 518개의 유전자중 Control 대비 3배 이상 증가되고 혈액에서 검출할 수 있는 soluble form의 단백질을 coding 하고 있는 4개(Tgfbi, Cst7, Angptl4 또는 Srgn)의 유전자를 선정한 바, 생물학적 시료에서 Tgfbi, Cst7, Angptl4 또는 Srgn의 발현 비교 분석을 통해 뇌미세혈관의 손상으로 인한 뇌 질환의 진단을 위한 정보를 제공할 수 있다. According to one embodiment of the present invention, in order to confirm the biological response over time after BBB opening according to focused ultrasound irradiation (1 hour, 6 hours, 12 hours, 24 hours, 48 hours) mild BBB degenerated brain tissue After obtaining, when the gene expression change over time was compared with the control group through RNA sequencing analysis, at 48 hours, about 518 genes increased and 174 genes showed a tendency to decrease, and among 518 genes The expression of Tgfbi, Cst7, Angptl4 or Srgn in biological samples was selected by selecting 4 genes (Tgfbi, Cst7, Angptl4 or Srgn) that are increased more than 3 times compared to the control and code for proteins in soluble form that can be detected in blood. Comparative analysis can provide information for the diagnosis of brain diseases caused by brain microvascular damage.
본 발명에 있어서, 진단을 위한 정보제공방법은 진단을 위한 예비적 단계로써 뇌미세혈관의 손상으로 인한 뇌 질환의 진단을 위하여 필요한 객관적인 기초정보를 제공하는 것이며 의사의 임상학적 판단 또는 소견은 제외된다. In the present invention, the method for providing information for diagnosis is a preliminary step for diagnosis, which provides objective basic information necessary for the diagnosis of brain diseases caused by damage to brain microvessels, and the clinical judgment or opinion of a doctor is excluded. .
본 발명에 있어서, '생물학적 시료'란 개체로부터 분리되어 목적 유전자 또는 단백질의 발현 수준을 측정하는 직접적인 대상을 의미하고, 조직, 세포, 전혈, 혈청, 혈장, 타액 또는 뇨와 같은 시료 등을 포함한다. 예를 들어, 본 발명의 시료란 뇌미세혈관의 손상으로 인한 뇌 질환 의심 개체에서 뇌 질환의 발병 여부를 진단하기 위한 시료로서, 바람직하게는 혈액일 수 있다. In the present invention, 'biological sample' refers to a direct target for measuring the expression level of a target gene or protein separated from an individual, and includes samples such as tissues, cells, whole blood, serum, plasma, saliva or urine . For example, the sample of the present invention is a sample for diagnosing the onset of a brain disease in a subject suspected of having a brain disease due to brain microvascular damage, and may preferably be blood.
상기 개체는 임의의 포유동물에 적용가능하며, 상기 포유동물은 인간 및 영장류뿐만 아니라, 소, 돼지, 양, 말, 개 및 고양이 등의 가축을 포함한다.The subject is applicable to any mammal, and the mammal includes humans and primates as well as livestock such as cattle, pigs, sheep, horses, dogs, and cats.
본 발명에 있어서, 'mRNA 발현 수준을 측정하기 위한 분석 방법'에는 중합효소반응(PCR), 역전사 중합효소반응 (RT-PCR), 경쟁적 역전사 중합효소반응(Competitive RT-PCR), 실시간 역전사 중합효소반응(Realtime RT-PCR), RNase 보호분석법(RPA; RNase protection assay), 노던 블랏팅(northern blotting), 또는 DNA 마이크로어레이 분석법 등이 있으나, 이에 제한되지 않는다.In the present invention, the 'analysis method for measuring mRNA expression level' includes polymerase reaction (PCR), reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase Reaction (Realtime RT-PCR), RNase protection assay (RPA; RNase protection assay), northern blotting, or DNA microarray analysis, but is not limited thereto.
본 발명에 있어서, '단백질 활성 수준을 측정하기 위한 분석 방법'에는 웨스턴 블랏팅(western blotting), ELISA(enzyme linked immunosorbent assay), 방사선면역분석법(Radioimmunoassay), 방사면역 확산법 (Radioimmunodiffusion), 오우크테로니(Ouchterlony) 면역 확산법, 로케트(Rocket) 면역전기영동, 조직면역염색, 면역 침전분석법(immunoprecipitation assay), 보체 고정 분석법(complete fixation assay), 유세포분석법 (Fluorescence Activated Cell Sorter, FACS) 또는 단백질 칩(protein chip) 분석법 등이 있으나, 이에 제한되지 않는다. In the present invention, the 'analysis method for measuring the protein activity level' includes western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay, radioimmunodiffusion, and Oktero Ouchterlony immunodiffusion assay, Rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complete fixation assay, Fluorescence Activated Cell Sorter (FACS) or protein chip ( protein chip) analysis method, etc., but is not limited thereto.
본 발명의 방법은 상기 측정된 Tgfbi, Cst7, Angptl4 또는 Srgn 유전자의 mRNA 발현 또는 이의 단백질 활성 수준을 대조군에서 측정된 수준과 비교하는 단계를 포함할 수 있다. 상기 대조군은 뇌미세혈관의 손상으로 인한 뇌 질환이 발명하지 않은 건강한 사람이다. 이에 따라 본 발명의 방법은 Tgfbi, Cst7, Angptl4 또는 Srgn 유전자의 mRNA 발현 또는 이의 단백질 활성 수준이 대조군 보다 높을 경우, 뇌미세혈관의 손상으로 인한 뇌 질환이 진행 중이거나 이미 발병하였을 것으로 판단 할 수 있다. The method of the present invention may include comparing the measured level of mRNA expression or protein activity of the Tgfbi, Cst7, Angptl4 or Srgn gene with the level measured in a control group. The control group was a healthy person who did not develop a brain disease due to brain microvascular damage. Accordingly, in the method of the present invention, when the mRNA expression of the Tgfbi, Cst7, Angptl4 or Srgn gene or the protein activity level thereof is higher than that of the control group, it can be determined that a brain disease caused by damage to brain microvessels is in progress or has already occurred. .
(a) 생물학적 샘플에 임의의 화합물을 처리하는 단계; (b) 상기 생물학적 샘플로부터 Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군으로부터 선택되는 어느 하나 이상의 유전자 또는 이의 단백질의 발현 수준 또는 활성 수준을 확인하는 단계; 및 (c) 상기 발현 수준을 임의의 화합물을 처리하지 않은 정상대조군에서의 상기 유전자 또는 이의 발현 단백질의 수준과 또는 활성 수준을 비교하는 단계; 를 포함하는 뇌미세혈관의 손상으로 인한 뇌 질환의 예방 또는 치료용 약학적 조성물을 스크리닝 하는 방법을 제공할 수 있다. (a) treating the biological sample with any compound; (b) confirming the expression level or activity level of any one or more genes or proteins thereof selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn from the biological sample; And (c) comparing the expression level with the level of the gene or its expression protein or activity level in a normal control group not treated with any compound; It is possible to provide a method for screening a pharmaceutical composition for the prevention or treatment of brain diseases caused by damage to brain microvessels comprising a.
본 발명에 있어서, 스크리닝은 뇌미세혈관의 손상으로 인한 뇌 질환의 치료 효과를 가지는 물질을 선별하는 조작에 관한 것이다. In the present invention, screening relates to an operation of selecting a substance having a therapeutic effect on brain diseases caused by damage to brain microvessels.
본 발명에 있어서, 상기 (a) 단계의 후보물질은 뇌 질환의 치료 활성을 테스트할 물질을 의미하며, 예컨대 추출물, 단백질, 올리고펩티드, 소형 유기 분자, 다당류, 폴리뉴클레오티드 및 광범위한 화합물 들의 임의 분자를 포함할 수 있다. 이러한 후보 물질은 천연물질뿐 아니라, 합성 물질도 포함한다. In the present invention, the candidate substance in step (a) means a substance to be tested for therapeutic activity of brain diseases, such as extracts, proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides, and any molecule of a wide range of compounds. can include These candidate materials include natural materials as well as synthetic materials.
본 발명에 있어서, 상기 (b) 단계의 Tgfbi, Cst7, Angptl4 또는 Srgn 유전자의 mRNA 발현 또는 이의 단백질 활성 수준을 측정하는 단계는 시료에서 후보물질 처리에 의한 Tgfbi, Cst7, Angptl4 또는 Srgn을 암호화하는 핵산 또는 Tgfbi, Cst7, Angptl4 또는 Srgn 단백질의 존재 여부와 발현 정도를 확인하는 과정이다. In the present invention, the step of measuring the mRNA expression or protein activity level of the Tgfbi, Cst7, Angptl4 or Srgn gene in step (b) is a nucleic acid encoding Tgfbi, Cst7, Angptl4 or Srgn by treatment with a candidate substance in the sample. Alternatively, it is a process of confirming the presence and expression level of Tgfbi, Cst7, Angptl4 or Srgn protein.
본 발명의 스크리닝 방법은 (c) 상기 (b) 단계에서 측정한 Tgfbi, Cst7, Angptl4 또는 Srgn 유전자의 mRNA 또는 이의 단백질의 활성이 상기 후보물질을 처리하지 않은 대조군의 Tgfbi, Cst7, Angptl4 또는 Srgn 유전자의 mRNA 발현 또는 이의 단백질의 활성보다 낮은 경우, 후보물질을 뇌 질환의 치료제로 판단하는 단계를 추가로 더 포함할 수 있다. 다시 말해, 후보물질의 처리에 의해 Tgfbi, Cst7, Angptl4 또는 Srgn 유전자의 mRNA 발현 또는 이의 단백질의 활성이 후보물질 비처리군의 Tgfbi, Cst7, Angptl4 또는 Srgn의 mRNA 발현 또는 이의 단백질의 활성보다 낮아지는 경우 상기 후보물질을 뇌 질환 치료제로 판단할 수 있고, 그와 반대로 Tgfbi, Cst7, Angptl4 또는 Srgn 유전자의 mRNA 발현 또는 이의 단백질의 활성이 후보물질 비처리군의 Tgfbi, Cst7, Angptl4 또는 Srgn 유전자의 mRNA 발현 또는 이의 단백질 활성량과 비슷하거나 보다 높아지는 경우 뇌미세혈관의 손상으로 인한 뇌 질환 치료제로 사용할 수 없다 판단 할 수 있다. The screening method of the present invention (c) the activity of the mRNA or protein of the Tgfbi, Cst7, Angptl4 or Srgn gene measured in step (b) is the Tgfbi, Cst7, Angptl4 or Srgn gene of the control group not treated with the candidate substance. When lower than the mRNA expression of or the activity of its protein, a step of determining the candidate substance as a therapeutic agent for brain disease may be further included. In other words, the mRNA expression of the Tgfbi, Cst7, Angptl4 or Srgn gene or the activity of the protein thereof by the treatment of the candidate substance is lower than the mRNA expression of the Tgfbi, Cst7, Angptl4 or Srgn gene or the activity of the protein of the candidate substance untreated group. In this case, the candidate substance can be judged as a brain disease treatment, and on the contrary, the mRNA expression of Tgfbi, Cst7, Angptl4 or Srgn gene or the activity of its protein is the mRNA of Tgfbi, Cst7, Angptl4 or Srgn gene in the candidate substance untreated group. If the expression or its protein activity is similar to or higher than that, it can be determined that it cannot be used as a treatment for brain diseases caused by damage to brain microvessels.
(a) 마우스에 집속 초음파를 0.25 내지 0.27 MPa로 10초 내지 230초 동안 처리는 단계를 포함하는 optimal BBB 변성 마우스 모델을 제작하는 방법을 제공할 수 있다. (a) a method for producing an optimal BBB degenerated mouse model comprising the step of treating the mouse with focused ultrasound at 0.25 to 0.27 MPa for 10 to 230 seconds may be provided.
본 발명은 (a) 마우스에 집속 초음파 0.42 내지 0.44 MPa로 10초 내지 230초 동안 처리하는 단계를 포함하는 mild BBB 변성 마우스 모델을 제작하는 방법을 제공할 수 있다. The present invention can provide a method for producing a mouse model with mild BBB degeneration, comprising the steps of (a) treating the mouse with focused ultrasound at 0.42 to 0.44 MPa for 10 to 230 seconds.
중복되는 내용은 본 명세서의 복잡성을 고려하여 생락하며, 본 명세서에서 달리 정의되지 않은 용어들은 본 발명이 속하는 분야에서 통상적으로 사용되는 의미를 갖는 것이다.Redundant content is omitted in consideration of the complexity of the present specification, and terms not defined otherwise in the present specification have meanings commonly used in the field to which the present invention belongs.
이하, 실시예를 통하여 본 발명을 보다 상세하게 설명한다. 그러나 하기 실시 예는 본 발명의 내용을 구체화하기 위한 것일 뿐 이에 의해 본 발명이 한정되는 것은 아니다. Hereinafter, the present invention will be described in more detail through examples. However, the following examples are only for specifying the contents of the present invention, and the present invention is not limited thereto.
[실시예 1][Example 1]
BBB변성 모델의 제작 Fabrication of BBB degeneration model
만성 퇴행성 뇌질환은 대부분의 경우 BBB의 손상으로 인해 그 결합이 느슨해져 있다. 이에 본 연구진들은 집속 초음파를 이용하여 BBB의 개통 유도하였다. 뇌질환의 BBB 변성 정도를 조절하여, 퇴행성 뇌질환 모델의 초기 BBB 손상 정도의 동물 모델과 퇴행성 뇌질환 모델의 중기 BBB 손상 정도의 동물 모델을 제작하였다. 8주령의 ICR mice를 이용하여 집속 초음파 유도 BBB 개통 동물 모델을 제작하였다. MRgFUS 기기를 이용한 초음파 처리는 FUS 기기(RK-100)를 사용하였으며, 도 1에 기기 구성의 개략도가 나타나 있다. MR 영상유도 집중초음파 시스템은 도1에 개략도가 나타나 있다. 시스템은 초음파 컨트롤 시스템과 초음파 조사 시스템 (MR 환경 호환)으로 이루어져 있다. 구체적으로 초음파 컨트롤 시스템은 초음파 변환기에 RF 파워를 전달할 수 있는 fuction generator와 RF amplifier로 이루어져 있으며 초음파 변환기에 전달되는 power를 실시간으로 측정할 수 있는 power meter로 구성되어 있다. 초음파 조사 시스템은 초음파 변환기, 초음파 변환기를 3차원으로 정밀 제어 할 수 있는 3축 제어부, water tank로 구성되어 MRI 장비 환경 내에서도 구동가능하게 설계되어 있다. MR 영상유도 집중초음파 시스템은 Bruker 9.4T MRI (BioSpec 94/20 USR - 9.4T, Bruker, MA, USA) 시스템과 연동되어 있다. 구체적인 실험 프로토콜은 초음파 영상 진단용 조영제인 미소기포(DEFINITY®)를 생리식염수에 1:50의 비율로 희석하여 0.012ml/s의 속도로 동물의 정맥에 주입한다. 희석된 미소기포를 주입하는 동안 1s에 10ms의 burst length를 가지는 집속 초음파를 MR영상 기반으로 원하는 뇌 지역에 2분간 조사하였다. MR 영상을 통해 MR 조영제 주입 전과 주입 후 영상을 비교하여 BBB 개폐를 확인할 수 있다. 뇌질환의 BBB 변성 정도에 따른 바이오 마커를 발굴하기 위해 optimal damage (0.25~0.27 MPa) 조건과 mild damage BBBD (0.42-0.44 MPa)조건의 초음파 에너지를 ICR mice의 뇌의 thalmus 부위에 조사하여 BBB 변성을 유도하였다. 초음파 조사조건은 중심주파수 1.1 MHz를 이용하여 RFP는 1Hz, 120초동안 조사하였다 (도 1). optimal damage (0.25~0.27 MPa) 조건과 mild damage BBBD (0.42-0.44 MPa)조건의 마우스의 MR영상을 확인하였다 (도 2). MR image는 BBB 개통후 조영제를 투여하고, 시간에 따른 enhancement 정도를 확인하였으며, 관상면(coronal), 축상면(axial) 그리고 시상면 (sagittal) 방향으로 이미지를 획득하였다. MR 조영제 투과정도에 따른 intensity 측정을 통해 optimal(~50%) 및 mild (~100%) BBB 조건으로 실험을 진행하였다. 집속초음파 조사에 따른 BBB 개통후 시간에 따른 생물학적 반응을 확인하기 위해서 시간에 따라 (1시간, 6시간, 12시간, 24시간, 48시간) BBB 변성 뇌조직을 획득하였으며, 각 그룹의 mouse에서 혈액을 약 1ml 채취하여 혈청을 분리하였다. In most cases of chronic degenerative brain disease, the connection between them is loose due to damage to the BBB. Therefore, the researchers induced opening of the BBB using focused ultrasound. By controlling the degree of BBB degeneration of brain diseases, an animal model of the degree of early BBB damage in the degenerative brain disease model and an animal model of the degree of BBB damage in the middle of the degenerative brain disease model were produced. An animal model for BBB patency induced by focused ultrasound was constructed using 8-week-old ICR mice. For ultrasonic treatment using the MRgFUS device, a FUS device (RK-100) was used, and a schematic diagram of the device configuration is shown in FIG. 1. The MR image-guided focused ultrasound system is schematically shown in FIG. The system consists of an ultrasonic control system and an ultrasonic irradiation system (compatible with MR environment). Specifically, the ultrasonic control system consists of a function generator and an RF amplifier that can deliver RF power to the ultrasonic transducer, and a power meter that can measure the power delivered to the ultrasonic transducer in real time. The ultrasonic irradiation system is composed of an ultrasonic transducer, a 3-axis controller that can precisely control the ultrasonic transducer in three dimensions, and a water tank, and is designed to be operable within the MRI equipment environment. The MR image-guided focused ultrasound system is linked with the Bruker 9.4T MRI (BioSpec 94/20 USR - 9.4T, Bruker, MA, USA) system. In the specific experimental protocol, microbubbles (DEFINITY®), a contrast agent for ultrasound imaging, are diluted in physiological saline at a ratio of 1:50 and injected into the vein of the animal at a rate of 0.012 ml/s. While injecting the diluted microbubbles, focused ultrasound with a burst length of 10 ms per 1 s was irradiated to the desired brain region based on MR images for 2 minutes. Through MR imaging, it is possible to confirm opening and closing of the BBB by comparing images before and after MR contrast agent injection. In order to discover biomarkers according to the degree of BBB degeneration of brain diseases, ultrasonic energy under optimal damage (0.25-0.27 MPa) and mild damage BBBD (0.42-0.44 MPa) conditions was irradiated to the thalmus of the brain of ICR mice to degenerate the BBB. induced. RFP was irradiated for 1 Hz and 120 seconds using a center frequency of 1.1 MHz as an ultrasonic irradiation condition (FIG. 1). MR images of mice under optimal damage (0.25-0.27 MPa) and mild damage BBBD (0.42-0.44 MPa) conditions were confirmed (FIG. 2). For MR images, after opening the BBB, a contrast agent was administered, the degree of enhancement over time was confirmed, and images were acquired in the coronal, axial, and sagittal directions. Experiments were conducted under optimal (~50%) and mild (~100%) BBB conditions through intensity measurement according to the degree of penetration of the MR contrast agent. In order to confirm the biological response over time after BBB opening according to focused ultrasound irradiation, BBB-modified brain tissue was obtained over time (1 hour, 6 hours, 12 hours, 24 hours, 48 hours), and blood samples were collected from each group of mice. About 1 ml was collected and serum was separated.
[실시예 2][Example 2]
Transcriptome analysis (전사체 분석) Transcriptome analysis
실시예 1의 방법으로 제작된 optimal damage BBB 동물 모델 및 mild damage BBB 동물모델에 집속 초음파를 조사한 뇌 부분의 조직을 액체 질소에 급속 냉동시킨 후, RNA isolation kit (Qiagen)를 이용하여 RNA를 분리하였다. 총 RNA를 Dnase를 처리하여 DNA 오염을 방지하고, library 제작을 위해 random fragmentation을 제작하였다. RNA fragment를 역전사 효소로 반응시켜 cDNA를 제작하고 만들어 cDNA 양쪽 끝에 adpator를 붙여 sequencing을 수행하였다. sequencing을 통해 얻어진 raw data의 quality를 분석하고 각 샘플의 transcript quantification을 통해 발현량을 FPKM(Fragments per kilobase of transcript per milion mapped reads)값으로 추출하였다 (도 3). 퇴행성 뇌질환의 초기단계로 예상할 수 있는 optimal BBBD 조건에서 RNA sequencing 분석을 통해 시간에 따른 유전자의 발현 변화를 control과 비교하였을 때, 약 20,000개의 유전자중 BBBD에 의해 증가 또는 감소되는 유전자의 발현차이가 약 30개에서 70개 정도로 control과 크게 차이가 없음을 확인 하였다. BBB의 손상이 중반 정도로 예상할 수 있는 mild damage BBBD 조건에서 RNA sequencing 분석 결과를 통해 시간에 따른 유전자 발현 변화를 control 그룹과 비교하였을 때, 48시간 째 약 518개의 유전자가 증가되었고 174개의 유전자가 감소되는 경향을 나타내었다 (도 3). 상기의 결과를 바탕으로 mild damage BBBD 조건에서의 혈액이나 뇌 조직 바이오 마커 후보군을 선정하였다. 518개의 유전자중 Control 대비 3배 이상 증가되고 혈액에서 검출할 수 있는 soluble form의 단백질을 coding 하고 있는 4개의 유전자를 선정하여 후보군으로 선정하였다 (도 4). After the optimal damage BBB animal model and the mild damage BBB animal model produced by the method of Example 1 were rapidly frozen in liquid nitrogen for the brain tissue irradiated with focused ultrasound, RNA was isolated using an RNA isolation kit (Qiagen). . Total RNA was treated with Dnase to prevent DNA contamination, and random fragmentation was produced for library production. The RNA fragment was reacted with reverse transcriptase to prepare cDNA, and adpators were attached to both ends of the cDNA to perform sequencing. The quality of raw data obtained through sequencing was analyzed, and the expression level was extracted as FPKM (Fragments per kilobase of transcript per milion mapped reads) value through transcript quantification of each sample (FIG. 3). Differences in expression of genes increased or decreased by BBBD among about 20,000 genes when comparing gene expression changes over time with control through RNA sequencing analysis under optimal BBBD conditions that can be expected in the early stage of degenerative brain disease It was confirmed that there was no significant difference from the control at about 30 to 70. When comparing gene expression changes over time with the control group through RNA sequencing analysis under mild damage BBBD conditions that can be expected to have moderate BBB damage, about 518 genes were increased and 174 genes were decreased at 48 hours showed a tendency to become (Fig. 3). Based on the above results, blood or brain tissue biomarker candidates under mild damage BBBD conditions were selected. Among the 518 genes, 4 genes coding for proteins in soluble form that can be detected in blood and increased by more than 3 times compared to the control were selected and selected as candidate groups (FIG. 4).
[실시예 3][Example 3]
유전체 분석 결과와 유전자 발현량의 상관관계 검증Verification of correlation between genome analysis results and gene expression levels
상기 실시예３의 후보 유전자의 sequencing 결과가 유의한지 확인하기 위해 real time PCR실험기법으로 후보군유전자 발현을 확인하여 상관관계를 확인하였다. 실시예 1의 방법으로 제작된 optimal damage BBB 동물 모델 및 mild damage BBB 동물모델에 집속 초음파를 조사한 뇌 부분의 조직을 액체 질소에 급속 냉동시킨 후, Quiagne RNA isolation kit (Quiagen)를 이용하여 RNA를 분리하였다. 총 RNA를 Dnase를 처리하여 DNA 오염을 방지하고, library 제작을 위해 random fragmentation을 제작하였다. RNA fragment를 역전사 효소로 반응시켜 cDNA를 제작하였다. SYBR® 그린 마스터 PCR 믹스 10 μL, 포워드 및 리버스 프라이머 각 5 pmol, 희석된 cDNA 주형 1 μL 및 적절한 양의 멸균 증류수를 함유한 20 μL 반응물 부피에서 SYBR® 그린 리얼-타임 PCR 키트를 사용하여 qRT-PCR을 수행했다. 유전자 증폭 조건은 다음과 같았다. 3 분 간 95 ℃에서 초기 변성; 15 초간 95 ℃에서 변성, 60 초간 60 ℃에서 아닐링 및 60 초간 72 ℃에서 신장 40 사이클; 및 5 분간 72 ℃에서 마지막 신장하였다. ABI 스텝 원 플러스 리얼-타임 PCR 시스템에서 qRT-PCR을 수행했다. 모든 정량은 내부 표준으로 U6과 함께 수행되었다. PCR 프라이머 서열은 하기 표 1과 같다. 그 결과 시간에 따른 유전자의 sequencing결과와 qRT-PCR결과가 유사하다는 것을 확인하였다 (도 4). In order to confirm that the sequencing results of the candidate genes in Example 3 were significant, the expression of the candidate genes was checked using real-time PCR testing technique to confirm the correlation. The tissue of the optimal damage BBB animal model and the mild damage BBB animal model prepared by the method of Example 1 were rapidly frozen in liquid nitrogen, and RNA was isolated using the Quiagne RNA isolation kit (Quiagen). did Total RNA was treated with Dnase to prevent DNA contamination, and random fragmentation was produced for library production. RNA fragments were reacted with reverse transcriptase to prepare cDNA. qRT-q using the SYBR® Green real-time PCR kit in a 20 μL reaction volume containing 10 μL of SYBR® Green master PCR mix, 5 pmol each of forward and reverse primers, 1 μL of diluted cDNA template, and an appropriate amount of sterile distilled water. PCR was performed. Gene amplification conditions were as follows. initial denaturation at 95 °C for 3 min; 40 cycles of denaturation at 95°C for 15 seconds, annealing at 60°C for 60 seconds, and elongation at 72°C for 60 seconds; and a final extension at 72° C. for 5 minutes. qRT-PCR was performed on an ABI Step One Plus real-time PCR system. All quantifications were performed with U6 as an internal standard. PCR primer sequences are shown in Table 1 below. As a result, it was confirmed that the gene sequencing results and the qRT-PCR results according to time were similar (FIG. 4).
| GeneGene | SequcneSequcne | Sequence numberSequence number |
| TGFBITGFBI | TggatgtcctgaagggagacTggatgtcctgaagggagac | 서열번호 1SEQ ID NO: 1 |
| GgcatttgccaagagtttgtGgcatttgccaagagtttgt | 서열번호 2SEQ ID NO: 2 | |
| Cst7Cst7 | AtgcatcaccaactggacaaAtgcatcaccaactggacaa | 서열번호 3SEQ ID NO: 3 |
| CagaggagaacaggcacctcCagaggagaacaggcacctc | 서열번호 4SEQ ID NO: 4 | |
| Angptl4Angptl4 | ActgccaggaactcttccaaActgccaggaactcttccaa | 서열번호 5SEQ ID NO: 5 |
| AtcactgtccagcctccatcAtcactgtccagcctccatc | 서열번호 6SEQ ID NO: 6 | |
| SrgnSrgn | TtttgatggaaggaccctcaTtttgatggaaggaccctca | 서열번호 7SEQ ID NO: 7 |
| TgttggctggtattcccattTgttggctggtattcccatt | 서열번호 8SEQ ID NO: 8 |
[실시예 4][Example 4]
ELISAELISA
집속초음파를 이용한 BBB 개통을 유도한 동물모델을 시간에 따라 sacrifice하고, 하대정맥에서 혈액을 약 1ml 정도 취하여, 상온에서 약 30분 혈액을 응고시킨 후, 6000rpm에서 30분간 원심 분리하여 상층액만을 분리하여 serum을 획득하였다. TGFBI Abcam 社 (ca. ab206987), CST7 R&D system 社 (ca. MSCTC0), Angptl4 LSBio 社 (Ca. LS-F6608), Srgn LSBio 社 (Ca. LS-F55170) ELISA kit를 이용하여 실험을 수행하기 위해서 serum을 50배와 10배로 blocking buffer에 희석 한 후에 ELISA plate에 100 ul씩 분주하였다. 상온에서 2시간동안 incubation후에 wash buffer로 5분씩 세 번 세척하였다. Biotin이 labelling되어 있는 antibody solution을 100ul씩 분주하고 37 ℃, 1시간동안 incubation하고, wash buffer로 5분씩 3번 세척하였다. ABC solution을 100ul씩 분주하고 37 ℃에서 30분 동안 incubation하고 wash buffer로 세척 후에 TMB substrate solution 90ul를 첨가하여 37 ℃에서 30분 동안 incubation후에 100ul의 stop solution을 첨가하고 plate reader (Tecan 社)를 이용하여 450nm 파장에서 흡광도를 측정하여 결과를 분석하였다. 혈액에서 Tgfbi의 발현은 0.42MPa에서 48시간째 두배 이상 증가되었다. Cst7의 발현은 0.25MPa 과 0.42MPa조건에서 24시간째 증가됨을 확인하였다. 혈액 내 Angpt14의 발현은 0.25MPa과 0.42MPa 조건에서 BBB 개통 후 1시간 이후부터 48시간까지 발현이 증가됨을 확인하였다. Srgn은 0.25MPa에서 12시간째 발현이 감소되었다가 48시간째 정상 수준으로 회복되었으며, 0.42MPa 조건에서는 24시간째 약 1.2배정도 증가됨을 확인하였다. 이를 통해 뇌미세혈관 손상에 의한 혈액 바이오 마커 발굴 가능성을 확인하였다. An animal model inducing BBB opening using focused ultrasound was sacrificed over time, about 1ml of blood was taken from the inferior vena cava, the blood was coagulated at room temperature for about 30 minutes, and only the supernatant was separated by centrifugation at 6000 rpm for 30 minutes serum was obtained. TGFBI Abcam (ca. ab206987), CST7 R&D system (ca. MSCTC0), Angptl4 LSBio (Ca. LS-F6608), Srgn LSBio (Ca. LS-F55170) To perform experiments using ELISA kits Serum was diluted 50-fold and 10-fold in blocking buffer, and then dispensed in 100 ul each on an ELISA plate. After incubation at room temperature for 2 hours, it was washed three times for 5 minutes each with wash buffer. Biotin-labeled antibody solution was dispensed by 100ul, incubated at 37 °C for 1 hour, and washed three times with wash buffer for 5 minutes each. After dispensing 100ul of ABC solution, incubating at 37 ℃ for 30 minutes, washing with wash buffer, adding 90ul of TMB substrate solution, incubating at 37 ℃ for 30 minutes, adding 100ul of stop solution and using a plate reader (Tecan) The absorbance was measured at a wavelength of 450 nm and the results were analyzed. The expression of Tgfbi in blood was more than doubled at 48 hours at 0.42 MPa. It was confirmed that the expression of Cst7 was increased at 24 hours under the conditions of 0.25 MPa and 0.42 MPa. It was confirmed that the expression of Angpt14 in the blood increased from 1 hour after BBB opening to 48 hours under the conditions of 0.25 MPa and 0.42 MPa. It was confirmed that the expression of Srgn decreased at 0.25 MPa at 12 hours and returned to normal at 48 hours, and increased by about 1.2 times at 24 hours at 0.42 MPa. Through this, the possibility of discovering blood biomarkers due to brain microvascular damage was confirmed.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far, the present invention has been looked at with respect to its preferred embodiments. Those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent scope will be construed as being included in the present invention.
Claims (11)
- Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군에서 선택된 적어도 어느 하나의 유전자를 포함하는, 뇌미세혈관의 손상으로 인한 뇌 질환 진단용 바이오 마커 조성물.A biomarker composition for diagnosing brain diseases caused by brain microvascular damage, comprising at least one gene selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn.
- Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군에서 선택된 적어도 어느 하나의 유전자의 mRNA 발현 수준 또는 이의 단백질 활성 수준을 측정하는 제제를 포함하는, 뇌미세혈관의 손상으로 인한 뇌 질환 진단용 조성물. Tgfbi, Cst7, Angptl4 and a composition for diagnosing brain diseases caused by damage to brain microvessels, comprising an agent for measuring the mRNA expression level or protein activity level of at least one gene selected from the group consisting of Srgn.
- 제2 항에 있어서, According to claim 2,상기 제제는 Tgfbi, Cst7, Angptl4 또는 Srgn의 유전자에 특이적으로 결합하는 프라이머 또는 프로브를 포함하는, 뇌미세혈관의 손상으로 인한 뇌 질환 진단용 조성물. The formulation is a composition for diagnosing brain diseases caused by damage to brain microvessels, including primers or probes that specifically bind to genes of Tgfbi, Cst7, Angptl4 or Srgn.
- 제2 항에 있어서, According to claim 2,상기 제제는 Tgfbi, Cst7, Angptl4 또는 Srgn의 유전자에 특이적으로 결합하는 항체인, 뇌미세혈관의 손상으로 인한 뇌 질환 진단용 조성물. The agent is an antibody that specifically binds to the gene of Tgfbi, Cst7, Angptl4 or Srgn, a composition for diagnosing brain diseases caused by damage to brain microvessels.
- 제2 항 내지 제4 항 중 어느 한 항의 조성물을 포함하는, 뇌미세혈관의 손상으로 인한 뇌 질환 진단용 키트. A kit for diagnosing brain diseases caused by damage to brain microvessels, comprising the composition of any one of claims 2 to 4.
- 생물학적 시료로부터 Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군에서 선택된 적어도 어느 하나의 유전자의 mRNA 발현 수준 또는 이의 단백질 활성 수준을 측정하는 단계;를 포함하는 뇌미세혈관의 손상으로 인한 뇌 질환 진단을 위한 정보제공방법. Measuring the mRNA expression level or protein activity level of at least one gene selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn from a biological sample; information for diagnosing brain diseases caused by damage to brain microvessels, including How to provide.
- (a) 생물학적 샘플에 임의의 화합물을 처리하는 단계; (a) treating the biological sample with any compound;(b) 상기 생물학적 샘플로부터 Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군에서 선택된 어느 하나의 유전자 또는 이의 단백질의 발현 수준 또는 활성 수준을 확인하는 단계; 및 (b) confirming the expression level or activity level of any one gene or protein thereof selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn from the biological sample; and(c) 상기 발현 수준을 임의의 화합물을 처리하지 않은 정상대조군에서의 상기 유전자 또는 이의 발현 단백질의 수준과 또는 활성 수준을 비교하는 단계를 포함하는 뇌미세혈관의 손상으로 인한 뇌 질환의 예방 또는 치료용 약학적 조성물을 스크리닝 하는 방법. (c) preventing or treating brain diseases caused by brain microvascular damage comprising comparing the expression level with the level of the gene or its expressed protein or activity level in a normal control group not treated with any compound A method for screening a pharmaceutical composition for use.
- (a) 마우스에 집속 초음파를 0.25 내지 0.27 MPa로 10초 내지 230초 동안 처리는 단계를 포함하는 optimal BBB 변성 마우스 모델을 제작하는 방법.(a) a method for producing an optimal BBB degeneration mouse model comprising the step of treating the mouse with focused ultrasound at 0.25 to 0.27 MPa for 10 seconds to 230 seconds.
- (a) 마우스에 집속 초음파 0.42 내지 0.44 MPa로 10초 내지 230초 동안 처리하는 단계를 포함하는 mild BBB 변성 마우스 모델을 제작하는 방법.(a) A method for producing a mild BBB degeneration mouse model comprising the step of treating the mouse with focused ultrasound at 0.42 to 0.44 MPa for 10 seconds to 230 seconds.
- 뇌미세혈관의 손상으로 인한 뇌 질환을 진단하기위한 Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군에서 선택된 적어도 어느 하나의 유전자를 포함하는 바이오 마커 조성물의 용도.Use of a biomarker composition containing at least one gene selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn for diagnosing brain diseases caused by brain microvascular damage.
- 생물학적 시료로부터 Tgfbi, Cst7, Angptl4 및 Srgn으로 이루어진 군에서 선택된 적어도 어느 하나의 유전자의 mRNA 발현 수준 또는 이의 단백질 활성 수준을 측정하는 단계;를 포함하는 뇌미세혈관의 손상으로 인한 뇌 질환의 진단 방법.Measuring the mRNA expression level or protein activity level of at least one gene selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn from a biological sample; A method for diagnosing brain diseases caused by damage to brain microvessels, comprising:
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